Genes

ABSTRACT

The invention relates to mutations in B-Raf gene products. The mutations described are identified in human tumours of natural origin. These mutations are associated with cancerous phenotypes and can be used as a basis for the diagnosis of cancer, cancerous cells or a predisposition to cancer in human subjects, and the development of anti-cancer therapeutics.

FIELD OF THE INVENTION

The present invention relates to cancer-specific mutants of B-raf genesand uses thereof in the detection of abnormal cells and cancer.Moreover, the invention describes methods for the diagnosis of cancer,the detection of cancerous cells in subjects and the development oftherapeutic agents for the treatment of cancer.

INTRODUCTION

Cancer can develop in any tissue of any organ at any age. Most cancersdetected at an early stage are potentially curable; thus, the ability toscreen patients for early signs of cancer, and thus allowing for earlyintervention, is highly desirable (See, for instance, the Merck Manualof Diagnosis and Therapy (1992) 16th ed., Merck & Co).

Cancerous cells display unregulated growth, lack of differentiation, andability to invade local tissues and metastasis. Thus cancer cells areunlike normal cells, and are potentially identifiable by not only theirphenotypic traits, but also by their biochemical and molecularbiological characteristics. Such characteristics are in turn dictated bychanges in cancerous cells which occur at the genetic level in a subsetof cellular genes known as oncogenes, which directly or indirectlycontrol cell growth and differentiation.

The Raf oncogene family includes three highly conserved genes termed A-,B- and C-raf (also called raf-1). C-Raf, the best characterised memberof the raf family, is the cellular homologue of v-raf, the transforminggene of the murine sarcoma virus 3611. The viral raf oncogene encodes aprotein that lacks the amino-terminal sequences of the normal Rafprotein. These amino-terminal sequences are crucial for the regulationof RAF serine/threonine-protein kinase activity, and their deletion orreplacement results in constitutive activity of the oncogene-encoded RAFprotein. This unregulated activity promotes cell proliferation,resulting in cell transformation. DNA from a few tumours has beenalleged to contain a transforming activity detectable by DNAtransfection of NIH/3T3 cells, identified as derived from truncatedC-raf-1. However, these results are likely to be transfection artefactsas the same mutations have not been found in the tumours from which thetransforming DNA was derived. Mutations created artificially in theC-raf gene, when introduced into cells in vitro can inducetransformation.

The B-raf gene is the human homologue of the avian c-Rmil protooncogeneencoding a 94-kD serine/threonine kinase detected in avian cells. Thisprotein contains amino-terminal sequences not found in other proteins ofthe mil/raf gene family. These sequences are encoded by 3 exons in theavian genome. Eychene et al. (1992) Oncogene 7:1657-1660 reported thatthese 3 exons are conserved in the human B-raf gene and that they encodean amino acid sequence similar to that of the avian gene. Theyidentified 2 human B-raf loci: B-raf 1, which was mapped to 7q34 byfluorescence in situ hybridisation and shown to encode the functionalgene product, and B-raf 2, an inactive processed pseudogene located onXq13.

By screening a mouse cDNA library with a v-raf oncogene probe, Huebneret al. (1986) Proc. Nat. Acad. Sci. 83: 3934-3938 isolated atransforming raf-related cDNA, A-raf, that represented a gene distinctfrom raft. The single A-raf locus of the mouse and the A-raf1 locus ofman are actively transcribed in several mouse and human cell lines. Thecomplete 606-amino acid sequence of the human A-raf1 oncogene has beendeduced from the 2,453-nucleotide sequence of the cDNA. The A-raf geneis X-linked.

A known mechanism for the conversion of proto-oncogenes to oncogenes isthe appearance of single mutations in the DNA sequence, known as pointmutations, which result in a change in the amino acid sequence of theencoded polypeptide. For example, ras oncogenes are not present innormal cells, but their proto-oncogene counterparts are present in allcells. The wild-type Ras proteins are small GTP-binding proteins thatare involved in signal transduction. However, many ras oncogenes fromviruses and human tumours have a point mutation in codon number 12: thecodon GGC that normally encodes a glycine is changed to GTC, whichencodes a valine. Multiple mutations have been documented at this codon,including at least 5 different substitutions which are activating. Thissingle amino acid change prevents the GTPase activity of the Rasprotein, and renders Ras constitutively activated, since it remainsGTP-bound. The amino acids at positions 13 and 61 are also frequentlychanged in ras oncogenes from human tumours.

The Raf protein is a serine/threonine kinase that is structurallyrelated to the protein kinase C (PKC) family, and is essential in cellgrowth and differentiation. Raf proteins are involved in signaltransduction in the activation of MAP kinase, which is highly conservedin eukaryotic organisms. MAP kinases (mitogen-activated proteinkinases), which include ERK1 and ERK2, directly phosphorylatetranscription factors to regulate biological events. MAPKKs (MAP kinasekinases) and MAPKKKs (MAPKK kinases) in turn regulate MAP kinases.

Raf proteins are MAPKKKs and are believed to phosphorylate the MAPKK MEKin vivo in mammalian biological systems. Distinct raf genes encodeA-Raf, B-Raf and Raf-1 (also known as c-Rat) in vertebrates (reviewed inPapin et al., 1998, Oncogene 12:2218-2221). The three proteins are notequal in their ability to activate MEK. A-Raf, the lesswell-characterised member of the family, appears to be a poor MEKactivator, its activity being difficult to measure (Pritchard et al.,1995, Mol. Cell. Biol. 15, 6430-6442). B-Raf and Raf-1 also differ intheir ability to activate MEK. While Raf-1 is ubiquitously expressed,B-Raf displays highest levels of expression in neural tissues (Barnieret al., 1995, J. Biol. Chem. 270, 23381-23389). However, B-Raf has beenidentified as the major MEK activator, even in cells where itsexpression is barely detectable by western blotting analysis (Catling etal., 1994; Jaiswal et al., 1994; Reuter et al., 1995; Huser et al.,2001; Mikula et al., 2001). Consistently, B-Raf displays higher affinityfor MEK-1 and MEK-2 than Raf-1 (Papin et al., 1996; Papin et al., 1998)and is more efficient in phosphorylating the MAPKK MEK.

The upstream activator of B-Raf is the GTPase Ras. A number of Rasisoforms are known to exist in mammals; N-Ras, Ha-Ras, Ki-Ras4A andKi-Ras4B. Other GTPases of the Ras superfamily may also interact withB-Raf. For example Rap-1, reviewed in Peysonnaux et al, (2001) Biologyof the Cell 93:53-62 appears to be a selective activator of B-Raf.

SUMMARY OF THE INVENTION

Point mutations in B-Raf gene products are described herein. The pointmutations described are identified in human tumours of natural origin.These point mutations are associated with cancerous phenotypes and canbe used as a basis for the diagnosis of cancer, cancerous cells or apredisposition to cancer in human subjects.

Since many of the signalling pathway(s) which are mediated by activationof the kinase activity of B-Raf are involved in control of cellproliferation and oncogenic transformation, it would be desirable to beable to rapidly detect changes in the B-raf gene which can result in anoncogenic character.

Thus, in a first aspect, there is provided a naturally-occurringcancer-associated mutant of a human B-Raf polypeptide comprising one ormore mutations.

Preferably, the cancer-associated mutant is isolated from anaturally-occurring primary human tumour.

Preferably, the mutation is in the kinase domain of B-Raf.

The present invention provides several such mutations, which have beenfound to be associated with a cancerous phenotype in human cancers; andthus establish a link between B-Raf mutations and cancer in vivo.

Preferably, the mutation is a point mutation. Mutations can also includechanges such as insertions, deletions or replacements of one or morethan one nucleotide, preferably of 2, 3, 4, 5 or 6 nucleotides.

Advantageously, the mutations are located C-terminal to amino acid 300in B-Raf. Preferred positions are 463, 465, 468, 585, 594, 595, 596 and599.

In a most preferred embodiment, the mutations are selected from thegroup consisting of V599E, V599D, G595R, G465V, G465E, G465A, G468A,G468E, E585K, F594L, G595R, L596V, L596R and G463E.

Preferably, the polypeptide is isolated.

The invention moreover encompasses fragments of the polypeptidesaccording to the invention, wherein said fragments include the mutationas described.

In a second aspect, there is provided a nucleic acid encoding a mutantB-Raf polypeptide or fragment thereof in accordance with the presentinvention. Preferably, the nucleic acid comprises one or more pointmutations.

Preferably, the nucleic acid is isolated.

Point mutations in B-raf genes have been detected which show associationwith tumours. Advantageously, the point mutation occurs at one or moreof positions 1388, 1394, 1403, 1753, 1782, 1783, 1796, 1797, 1787 and1786 of B-raf. Preferably, the point mutation is G1388T, G1783C,TG1796-97AT, G1394T, G1394A, G1394C, G1403C, G1403A, G1753A, T1782G,G1388A, T1796A, T1787G or C1786G in B-raf. The invention moreoverprovides the complement of any nucleic acid described above.

In a further embodiment, there is provided a nucleic acid whichhybridises specifically to a nucleic acid according to the invention, asdescribed herein. Such a nucleic acid can for example be a primer whichdirects specific amplification of a mutant B-Raf-encoding nucleic acidaccording to the invention in a nucleic acid amplification reaction.

In a third aspect, the invention provides a ligand which bindsselectively to a mutant B-Raf polypeptide according to the invention.

Such a ligand is advantageously an immunoglobulin, and is preferably anantibody or an antigen-binding fragment thereof.

According to a fourth aspect, there is provided a method for thedetection of cellular transformation comprising the steps of:

-   -   (a) isolating a sample of cellular material from a subject;    -   (b) examining nucleic acid material from at least part of one or        more B-raf genes in said cellular material; and    -   (c) determining whether such nucleic acid material comprises one        or more mutations in a sequence encoding a B-Raf polypeptide.

Advantageously, the mutation is a point mutation.

Advantageously, the mutation occurs at one or more of positions 1388,1394, 1403, 1753, 1782, 1783, 1796, 1797, 1787 and 1786 of B-raf.Preferably, the point mutation is G1388T, G1783C, TG1796-97AT, G1394T,G1394A, G1394C, G1403C, G1403A, G1753A, T1782G, G1388A, T1796A, T1787Gor C1786G in B-raf.

The mutations identified in accordance with the invention areadvantageously somatic mutations, which have occurred in somatic tissueand are not transmitted through the germ line. Thus, the inventionmoreover relates to a method for the detection of cellulartransformation, comprising the steps of:

-   -   (a) isolating a first sample of cellular material from a tissue        of a subject which is suspected to be cancerous, and a second        sample of cellular material from a non-cancerous tissue of the        same subject;    -   (b) examining nucleic acid material from at least part of one or        more B-raf genes in both said samples of cellular material; and    -   (c) determining whether such nucleic acid material comprises one        or more point mutations in a sequence encoding a B-Raf        polypeptide; and said mutation being present in the cellular        material from the suspected cancerous tissue but not present in        the cellular material from the non-cancerous tissue.

The invention moreover provides a method for the detection of cellulartransformation, comprising the steps of:

-   -   (a) obtaining a sample of cellular material from a subject;    -   (b) screening said sample with a ligand which binds selectively        to a mutant B-Raf polypeptide according to the invention; and    -   (c) detecting one or more mutant B-Raf polypeptides in said        sample.

In a still further aspect, the invention relates to a method foridentifying one or more compounds having anti-proliferative activity,comprising the steps of:

-   -   (a) providing one or more mutant B-Raf polypeptides in        accordance with the present invention;    -   (b) contacting said polypeptide(s) with one or more compounds to        be tested; and    -   (c) detecting an interaction between said one or more compounds        and said mutant polypeptides.

Preferably, the interaction is a binding interaction.

Moreover, the invention provides an assay for identifying one or morecompounds having anti-proliferative activity, comprising the steps of:

-   -   (a) providing one or more mutant B-Raf polypeptides in        accordance with the present invention;    -   (b) providing a downstream substrate for the B-Raf polypeptide;    -   (c) detecting modification of the substrate in presence of the        compound(s) to be tested.

B-Raf is a protein kinase, and accordingly substrates therefore arecapable of being phosphorylated or dephosphorylated. Preferably, theaction of mutant B-Raf on the substrate results in a detectable changetherein. Advantageously, the substrate is a further kinase orphosphatase, which in turn modifies a third molecule in which adetectable change occurs.

For example, the substrate may be the kinase MEK. MEK phosphorylationmay be detected directly, or, preferably, is detected through activationof MEK to phosphorylate MAP Kinase.

Advantageously, a reference activity of mutant B-Raf on the substrate isestablished, and the activity in the presence and/or absence of thecompound(s) to be tested compared to the reference value. A decrease inthe activity of the mutant B-Raf is indicative of a reduction inproliferative activity.

The invention moreover provides a cell-based assay for screeningcompounds for anti-proliferative activity. In a first embodiment, a theinvention provides a 3T3 focus-forming assay comprising the steps of:

-   -   (a) providing a culture of NIH 3T3 cells;    -   (b) transfecting said cells with a mutant B-raf nucleic acid in        accordance with the invention;    -   (c) exposing the cells to one or more compound(s) to be tested;        and    -   (d) determining the difference in the number of foci formed        between transfected cells exposed to said compound(s) to be        tested and transfected cells not so exposed.

The cell-based assay is commonly performed using NIH 3T3 cells. However,other cell types, especially fibroblast cells, can be used in such anassay.

Advantageously, a reference focus-forming activity of a mutant B-rafgene on the cells used in the assay is established, and the activity inthe presence and/or absence of the compound(s) to be tested compared tothe reference value. A decrease in the focus-forming activity of themutant B-raf gene is indicative of a reduction in proliferative activityand thus of antiproliferative activity in the compound(s) being tested.

Automated methods and apparata for the detection of mutations inaccordance with the invention are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: B-Raf activity assays. The kinase activity of B-Raf wasmeasured in an immunoprecipitation kinase cascade assay, using MBP asthe final substrate. The activity is shown as number of countsincorporated into MBP. The assay was performed in triplicate and theaverage is shown, with error bars to represent deviations from the mean.Both the basal kinase activity (open bars) and the ^(V12)Ras stimulatedkinase activities (hatched bars) are shown.

FIG. 1B: B-Raf activity assays. The kinase activity of B-Raf wasmeasured in an immunoprecipitation kinase cascade assay, using MBP asthe final substrate. The activity is shown as number of countsincorporated into MBP. The assay was performed in triplicate and theaverage is shown, with error bars to represent deviations from the mean.Both the basal kinase activity (open bars) and the ^(V12)Ras stimulatedkinase activities are shown.

FIG. 2: Transformation of NIH3T3 cells by B-Raf and activating mutants.The cells were transfected with the indicated constructs and the numberof colonies was determined. The results are the average of at leastthree assays. The number of colonies relative to the number induced byB-Raf is shown.

FIG. 3A. V599D is an activating mutation in BRAF. BRAF or ^(V599D)BRAFwere expressed alone, or together with oncogenic Ras as indicated. Theactivity of the BRAF proteins were determined using animmunoprecipitation kinase cascade assay in which immunoprecipitatedBRAF is used to sequentially activate MEK and ERK. The activation of ERKis determined using myelin basic protein and [³²P]-γATP as substrates.

FIG. 3B. Inhibition of ERK in melanoma cell lines using pharmacologicalreagents. WM266.4 or A375P cells were treated with 10 μM U0126, 10 μMBAY 43-9006 or DMSO as a control. Equivalent amounts of cellularproteins were resolved on SDS-gels and the levels of active ERK weredetermined using the ppERK antibody.

FIG. 4. Inhibition of cell growth by pharmacological agents. WM-266.4cells were incubated in the presence of U0126 (10 μM) or BAY 43-9006 (10μM) or the vehicle control (DMSO). After 48 hours, DNA synthesis wasdetermined by incubating the cells with [³H]-thymidine and the levels ofthymidine incorporated into the cellular DNA was determined.

FIG. 5A. CRAF expression is suppressed by siRNA. WM-266.4, Colo 829 orBE cells were treated with a CRAF specific siRNA probe (CRAF), thescrambled siRNA probe (scrambled), oligofectamine (oligo) or untreated(control). The cells were incubated for 24 hours and the levels of CRAFprotein was determined by Western blotting.

FIG. 5B. BRAF expression is suppressed by siRNA. WM-266.4, Colo 829 orBE cells were treated with a BRAF specific siRNA probe, the scrambledsiRNA probe, or were left untreated as shown. The cells were incubatedfor 24 hours and the levels of BRAF activity were tested using animmunoprecipitation kinase assay MEK and ERK as sequential assays. Theactivity of ERK was determined using MBP and [³²P]-γATP as substrates.

FIG. 6. Ablation of BRAF, but not CRAF blocks ERK activity in melanomacells. WM-266.4 or Colo 829 cells were treated with a BRAF specificsiRNA probe (BRAF), or the scrambled control (sBRAF), or a CRAF specificprobe (CRAF), or its scrambled control (sCRAF), or oligofectamine(oligo) or left untreated (control) as indicated. The cells wereincubated for the times indicated, and the Colo 829 cells were treatedfor 24 hours. The levels of ERK activity in equivalent amounts of cellextract was determined by Western blotting with the ppERK antibody.

FIG. 7. Ablation of BRAF, but not CRAF induces apoptosis in melanomacells. WM-266.4 cells were treated with a BRAF specific siRNA probe(BRAF), or the scrambled control (sBRAF), or a CRAF specific siRNA probe(CRAF), or U0126, or DMSO (oligo) or left untreated (control) asindicated. The cells were incubated for 96 hours and the cell cycleprofile was analysed by FACS, or PARP expression was examined by Westernblotting.

FIG. 8. B-Raf and GST-MKKI activity validation. Assay performed usingWTS1 B-Raf lysate (Batch A), GST MKK1 (6.5 μg/ml) and ERK2 (kinasecompetent, 100 μg/ml) to measure ³³P-γ-phosphate incorporation into MBP(0.3 mg/ml). Data shown are mean±SD of triplicate determinations.

FIG. 9. Assessment of Filter Plate and FlashPlate Radiometric AssayPlatform. Assay performed using WTS1 B-Rafllysate (Batch A) and GST MKK1(6.5 μg/ml) to measure ³³P-γ-phosphate incorporation into GST-kdERK2(100 μg/ml). Data shown are mean±SD of triplicate determinations.

FIG. 10. Assessment of DELFIA non-radiometric assay platform. 100 ngGST-kdERK2 was pre-bound to each well followed by the addition of B-Raflysate (Batch A), GST-MKK1 (6.5 μg/ml) and ATP (500 μM). Data shown aremean±SD of triplicate determinations.

FIG. 11. Titration of anti-phospho-ERK2 in DELFIA Assay. 100 ngGST-kdERK2 was pre-bound to the well followed by the addition of B-Raflysate (Batch A), GST-MKK1 (6.5 μg/ml) and ATP (500 μM). Data shown aremean of duplicate determinations.

FIG. 12. Titration of Europium-labelled Secondary antibody in DELFIAAssay. 100 ng GST-kdERK2 was pre-bound to the well followed by theaddition of B-Raf lysate (Batch A), GST-MKK1 (6.5 μg/ml) and ATP (500μM). Data shown are mean of duplicate determinations.

FIG. 13. Assessment of Homogenous Assay Protocol. The homogenous assaywas performed in a 96-well plate using a 50 μl reaction volumecontaining a B-Raf lysate 9Batch B), 6.5 μg/ml GST-MKK1, 80 μg/mlGST-kdERK2 and 500 μM ATP. Data shown are mean±SD of triplicatedeterminations.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridisation techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods. See,generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)Ed, John Wiley & Sons, Inc.; as well as Guthrie et al., Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Vol. 194,Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods andApplications (Innis, et al. 1990. Academic Press, San Diego, Calif.),McPherson et al., PCR Volume 1, Oxford University Press, (1991), Cultureof Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney.1987. Liss, Inc. New York, N.Y.), and Gene Transfer and ExpressionProtocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc.,Clifton, N.J.). These documents are incorporated herein by reference.

DEFINITIONS

The present application describes B-Raf polypeptide mutants. As usedherein, the term “RAF polypeptide” is used to denote a polypeptide ofthe RAF family. RAF was first identified in a cloned unique acutelytransforming replication-defective mouse type C virus, which containedan oncogene v-raf (Rapp, et al. Proc. Nat. Acad. Sci. 80: 4218-4222,1983). The cellular homologue, c-raf, is present in mammalian DNA. Otherhomologues have since been discovered in humans and birds, where raf hasbeen shown to be the homologue of the avian oncogene mil. B-Raf isrelated to RAF, but possesses three additional N-terminal exons. Theterm “B-Raf” thus encompasses all known human B-Raf homologues andvariants, as well as other polypeptides which show sufficient homologyto B-Raf to be identified as B-Raf homologues. The term does not includeARAF, CRAF or RAF1. Preferably, B-Raf is identified as a polypeptidehaving the sequence shown at accession no. NP_(—)004324, nucleic acidaccession no. NM_(—)004333. The term “B-Raf” preferably includespolypeptides which are 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous toNP_(—)004324. Homology comparisons can be conducted by eye, or moreusually, with the aid of readily available sequence comparison programs.These commercially available computer programs can calculate percentage(%) homology between two or more sequences.

Percentage homology can be calculated over contiguous sequences, i.e.one sequence is aligned with the other sequence and each amino acid inone sequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). It is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

A “fragment” of a polypeptide in accordance with the invention is apolypeptide fragment which encompasses the mutant amino acid(s)described in accordance with the invention. The fragment can be anylength up to the full length of B-Raf polypeptide; it thus encompassesB-Raf polypeptides which have been truncated by a few amino acids, aswell as shorter fragments. Advantageously, fragments are between about764 and about 5 amino acids in length; preferably about 5 to about 20amino acids in length; advantageously, between about 10 and about 50amino acids in length. Fragments according to the invention are useful,inter alia, for immunisation of animals to raise antibodies. Thus,fragments of polypeptides according to the invention advantageouslycomprise at least one antigenic determinant (epitope) characteristic ofmutant B-Raf as described herein. Whether a particular polypeptidefragment retains such antigenic properties can readily be determined byroutine methods known in the art. Peptides composed of as few as sixamino acid residues ore often found to evoke an immune response.

A “nucleic acid” of the present invention is a nucleic acid whichencodes a human B-Raf polypeptide as described above. The term moreoverincludes those polynucleotides capable of hybridising, under stringenthybridisation conditions, to the naturally occurring nucleic acidsidentified above, or the complement thereof. “Stringent hybridisationconditions” refers to an overnight incubation at 42° C. in a solutioncomprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulphate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C.

Although nucleic acids, as referred to herein, are generally naturalnucleic acids found in nature, the term can include within its scopemodified, artificial nucleic acids having modified backbones or bases,as are known in the art.

A nucleic acid encoding a fragment according to the invention can be theresult of nucleic acid amplification of a specific region of a B-rafgene, incorporating a mutation in accordance with the present invention.

An “isolated” polypeptide or nucleic acid, as referred to herein, refersto material removed from its original environment (for example, thenatural environment in which it occurs in nature), and thus is alteredby the hand of man from its natural state. For example, an isolatedpolynucleotide could be part of a vector or a composition of matter, orcould be contained within a cell, and still be “isolated” because thatvector, composition of matter, or particular cell is not the originalenvironment of the polynucleotide. Preferably, the term “isolated” doesnot refer to genomic or cDNA libraries, whole cell total or mRNApreparations, genomic DNA preparations (including those separated byelectrophoresis and transferred onto blots), sheared whole cell genomicDNA preparations or other compositions where the art demonstrates nodistinguishing features of the polypeptides/nucleic acids of the presentinvention.

The polypeptides according to the invention comprise one or moremutations. “Mutations” includes amino acid addition, deletion orsubstitution; advantageously, it refers to amino acid substitutions.Such mutations at the polypeptide level are reflected at the nucleicacid level by addition, deletion or substitution of one or morenucleotides. Generally, such mutations do not alter the reading frame ofthe nucleic acid. Advantageously, the changes at the nucleic acid levelare point mutations, in which a single nucleotide is substituted foranother, altering the codon of which it is part to specify a differentamino acid.

The mutations in B-Raf identified in the present invention occurnaturally, and have not been intentionally induced in cells or tissue bythe application of carcinogens or other tumorigenic factors. Thus, themutations identified herein accurately reflect natural tumorigenesis inhuman tissues to in vivo. Their detection is thus a far better basis fordiagnosis than the detection of mutations identified in rodents afterartificial chemical tumour induction.

A “somatic” mutation is a mutation which is not transmitted through thegerm line of an organism, and occurs in somatic tissues thereof.Advantageously, a somatic mutation is one which is determined to besomatic though normal/tumour paired sample analysis.

All amino acid and nucleotide numbering used herein starts from aminoacid+1 of the B-Raf polypeptide or the first ATG of the nucleotidesequence encoding it.

“Amplification” reactions are nucleic acid reactions which result inspecific amplification of target nucleic acids over non-target nucleicacids. The polymerase chain reaction (PCR) is a well known amplificationreaction.

An “immunoglobulin” is one of a family of polypeptides which retain theimmunoglobulin fold characteristic of immunoglobulin (antibody)molecules, which contains two β sheets and, usually, a conserveddisulphide bond. Members of the immunoglobulin superfamily are involvedin many aspects of cellular and non-cellular interactions in vivo,including widespread roles in the immune system (for example,antibodies, T-cell receptor molecules and the like), involvement in celladhesion (for example the ICAM molecules) and intracellular signalling(for example, receptor molecules, such as the PDGF receptor). Thepresent invention is preferably applicable to antibodies, which arecapable of binding to target antigens with high specificity.

“Antibodies” can be whole antibodies, or antigen-binding fragmentsthereof. For example, the invention includes fragments such as Fv andFab, as well as Fab′ and F(ab′)₂, and antibody variants such as scFv,single domain antibodies, Dab antibodies and other antigen-bindingantibody-based molecules.

“Cancer” is used herein to refer to neoplastic growth arising fromcellular transformation to a neoplastic phenotype. Such cellulartransformation often involves genetic mutation; in the context of thepresent invention, transformation involves genetic mutation byalteration of one or more B-raf genes as described herein.

Methods for Detection of Nucleic Acids

The detection of mutant nucleic acids encoding B-Raf can be employed, inthe context of the present invention, to diagnose the presence orpredisposition to cellular transformation and cancer. Since mutations inB-raf genes generally occur at the DNA level, the methods of theinvention can be based on detection of mutations in genomic DNA, as wellas transcripts and proteins themselves. It can be desirable to confirmmutations in genomic DNA by analysis of transcripts and/or polypeptides,in order to ensure that the detected mutation is indeed expressed in thesubject.

Mutations in genomic nucleic acid are advantageously detected bytechniques based on mobility shift in amplified nucleic acid fragments.For instance, Chen et al., Anal Biochem 1996 Jul. 15; 239 (1):61-9,describe the detection of single-base mutations by a competitivemobility shift assay. Moreover, assays based on the technique ofMarcelino et al., BioTechniques 26 (6): 1134-1148 (June 1999) areavailable commercially.

In a preferred example, capillary heteroduplex analysis may be used todetect the presence of mutations based on mobility shift of duplexnucleic acids in capillary systems as a result of the presence ofmismatches.

Generation of nucleic acids for analysis from samples generally requiresnucleic acid amplification. Many amplification methods rely on anenzymatic chain reaction (such as a polymerase chain reaction, a ligasechain reaction, or a self-sustained sequence replication) or from thereplication of all or part of the vector into which it has been cloned.Preferably, the amplification according to the invention is anexponential amplification, as exhibited by for example the polymerasechain reaction.

Many target and signal amplification methods have been described in theliterature, for example, general reviews of these methods in Landegren,U., et al., Science 242:229-237 (1988) and Lewis, R., GeneticEngineering News 10:1, 54-55 (1990). These amplification methods can beused in the methods of our invention, and include polymerase chainreaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligasehybridisation, Qbeta bacteriophage replicase, transcription-basedamplification system (TAS), genomic amplification with transcriptsequencing (GAWTS), nucleic acid sequence-based amplification (NASBA)and in situ hybridisation. Primers suitable for use in variousamplification techniques can be prepared according to methods known inthe art.

Polymerase Chain Reaction (PCR)

PCR is a nucleic acid amplification method described inter alia in U.S.Pat. Nos. 4,683,195 and 4,683,202. PCR consists of repeated cycles ofDNA polymerase generated primer extension reactions. The target DNA isheat denatured and two oligonucleotides, which bracket the targetsequence on opposite strands of the DNA to be amplified, are hybridised.These oligonucleotides become primers for use with DNA polymerase. TheDNA is copied by primer extension to make a second copy of both strands.By repeating the cycle of heat denaturation, primer hybridisation andextension, the target DNA can be amplified a million fold or more inabout two to four hours. PCR is a molecular biology tool, which must beused in conjunction with a detection technique to determine the resultsof amplification. An advantage of PCR is that it increases sensitivityby amplifying the amount of target DNA by 1 million to 1 billion fold inapproximately 4 hours. PCR can be used to amplify any known nucleic acidin a diagnostic context (Mok et al., (1994), Gynaecologic Oncology, 52:247-252).

Self-Sustained Sequence Replication (3SR)

Self-sustained sequence replication (3SR) is a variation of TAS, whichinvolves the isothermal amplification of a nucleic acid template viasequential rounds of reverse transcriptase (RT), polymerase and nucleaseactivities that are mediated by an enzyme cocktail and appropriateoligonucleotide primers (Guatelli et al. (1990) Proc. Natl. Acad. Sci.USA 87:1874). Enzymatic degradation of the RNA of the RNA/DNAheteroduplex is used instead of heat denaturation. RNase H and all otherenzymes are added to the reaction and all steps occur at the sametemperature and without further reagent additions. Following thisprocess, amplifications of 10⁶ to 10⁹ have been achieved in one hour at42° C.

Ligation Amplification (LAR/LAS)

Ligation amplification reaction or ligation amplification system usesDNA ligase and four oligonucleotides, two per target strand. Thistechnique is described by Wu, D. Y. and Wallace, R. B. (1989) Genomics4:560. The oligonucleotides hybridise to adjacent sequences on thetarget DNA and are joined by the ligase. The reaction is heat denaturedand the cycle repeated.

Replicase

In this technique, RNA replicase for the bacteriophage Qβ, whichreplicates single-stranded RNA, is used to amplify the target DNA, asdescribed by Lizardi et al. (1988) Bio/Technology 6:1197. First, thetarget DNA is hybridised to a primer including a T7 promoter and a Qβ 5′sequence region. Using this primer, reverse transcriptase generates acDNA connecting the primer to its 5′ end in the process. These two stepsare similar to the TAS protocol. The resulting heteroduplex is heatdenatured. Next, a second primer containing a Qβ 3′ sequence region isused to initiate a second round of cDNA synthesis. This results in adouble stranded DNA containing both 5′ and 3′ ends of the Qβbacteriophage as well as an active T7 RNA polymerase binding site. T7RNA polymerase then transcribes the double-stranded DNA into new RNA,which mimics the Qβ. After extensive washing to remove any unhybridisedprobe, the new RNA is eluted from the target and replicated by Qβreplicase. The latter reaction creates 10⁷ fold amplification inapproximately 20 minutes.

Alternative amplification technology can be exploited in the presentinvention. For example, rolling circle amplification (Lizardi et al.,(1998) Nat Genet. 19:225) is an amplification technology availablecommercially (RCAT™) which is driven by DNA polymerase and can replicatecircular oligonucleotide probes with either linear or geometric kineticsunder isothermal conditions.

In the presence of two suitably designed primers, a geometricamplification occurs via DNA strand displacement and hyperbranching togenerate 10¹² or more copies of each circle in 1 hour.

If a single primer is used, RCAT generates in a few minutes a linearchain of thousands of tandemly linked DNA copies of a target covalentlylinked to that target.

A further technique, strand displacement amplification (SDA; Walker etal., (1992) PNAS (USA) 80:392) begins with a specifically definedsequence unique to a specific target. But unlike other techniques whichrely on thermal cycling, SDA is an isothermal process that utilises aseries of primers, DNA polymerase and a restriction enzyme toexponentially amplify the unique nucleic acid sequence.

SDA comprises both a target generation phase and an exponentialamplification phase.

In target generation, double-stranded DNA is heat denatured creating twosingle-stranded copies. A series of specially manufactured primerscombine with DNA polymerase (amplification primers for copying the basesequence and bumper primers for displacing the newly created strands) toform altered targets capable of exponential amplification.

The exponential amplification process begins with altered targets(single-stranded partial DNA strands with restricted enzyme recognitionsites) from the target generation phase.

An amplification primer is bound to each strand at its complementary DNAsequence. DNA polymerase then uses the primer to identify a location toextend the primer from its 3′ end, using the altered target as atemplate for adding individual nucleotides. The extended primer thusforms a double-stranded DNA segment containing a complete restrictionenzyme recognition site at each end.

A restriction enzyme is then bound to the double stranded DNA segment atits recognition site. The restriction enzyme dissociates from therecognition site after having cleaved only one strand of thedouble-sided segment, forming a nick. DNA polymerase recognises the nickand extends the strand from the site, displacing the previously createdstrand. The recognition site is thus repeatedly nicked and restored bythe restriction enzyme and DNA polymerase with continuous displacementof DNA strands containing the target segment.

Each displaced strand is then available to anneal with amplificationprimers as above. The process continues with repeated nicking, extensionand displacement of new DNA strands, resulting in exponentialamplification of the original DNA target.

Once the nucleic acid has been amplified, a number of techniques areavailable for detection of single base pair mutations. One suchtechnique is Single Stranded Conformational Polymorphism (SSCP). SCCPdetection is based on the aberrant migration of single stranded mutatedDNA compared to reference DNA during electrophoresis. Mutation producesconformational change in single stranded DNA, resulting in mobilityshift. Fluorescent SCCP uses fluorescent-labelled primers to aiddetection. Reference and mutant DNA are thus amplified using fluorescentlabelled primers. The amplified DNA is denatured and snap-cooled toproduce single stranded DNA molecules, which are examined bynon-denaturing gel electrophoresis.

Chemical mismatch cleavage (CMC) is based on the recognition andcleavage of DNA mismatched base pairs by a combination of hydroxylamine,osmium tetroxide and piperidine. Thus, both reference DNA and mutant DNAare amplified with fluorescent labelled primers. The amplicons arehybridised and then subjected to cleavage using Osmium tetroxide, whichbinds to an mismatched T base, or Hydroxylamine, which binds tomismatched C base, followed by Piperidine which cleaves at the site of amodified base. Cleaved fragments are then detected by electrophoresis.

Techniques based on restriction fragment polymorphisms (RFLPs) can alsobe used. Although many single nucleotide polymorphisms (SNPs) do notpermit conventional RFLP analysis, primer-induced restriction analysisPCR (PIRA-PCR) can be used to introduce restriction sites using PCRprimers in a SNP-dependent manner. Primers for PIRA-PCR which introducesuitable restriction sites can be designed by computational analysis,for example as described in Xiaiyi et al., (2001) Bioinformatics17:838-839.

In an alternative embodiment, the present invention provides for thedetection of gene expression at the RNA level. Typical assay formatsutilising ribonucleic acid hybridisation include nuclear run-on assays,RT-PCR and RNase protection assays (Melton et al., Nuc. Acids Res.12:7035. Methods for detection which can be employed include radioactivelabels, enzyme labels, chemiluminescent labels, fluorescent labels andother suitable labels.

RT-PCR is used to amplify RNA targets. In this process, the reversetranscriptase enzyme is used to convert RNA to complementary DNA (cDNA),which can then be amplified using PCR. This method has proven useful forthe detection of RNA viruses. Its application is otherwise as for PCR,described above.

Methods for Detection of Polypeptides

The invention provides a method wherein a protein encoded a mutant B-rafgene is detected. Proteins can be detected by protein gel assay,antibody binding assay, or other detection methods known in the art.

For example, therefore, mutant B-Raf polypeptides can be detected bydifferential mobility on protein gels, or by other size analysistechniques such as mass spectrometry, in which the presence of mutantamino acids can be determined according to molecular weight. Peptidesderived from mutant B-Raf polypeptides, in particular, as susceptible todifferentiation by size analysis.

Advantageously, the detection means is sequence-specific, such that aparticular point mutation can accurately be identified in the mutantB-Raf polypeptide. For example, polypeptide or RNA molecules can bedeveloped which specifically recognise mutant B-Raf polypeptides in vivoor in vitro.

For example, RNA aptamers can be produced by SELEX. SELEX is a methodfor the in vitro evolution of nucleic acid molecules with highlyspecific binding to target molecules. It is described, for example, inU.S. Pat. Nos. 5,654,151, 5,503,978, 5,567,588 and 5270163, as well asPCT publication WO 96/38579, each of which is specifically incorporatedherein by reference.

The SELEX method involves selection of nucleic acid aptamers,single-stranded nucleic acids capable of binding to a desired target,from a library of oligonucleotides. Starting from a library of nucleicacids, preferably comprising a segment of randomised sequence, the SELEXmethod includes steps of contacting the library with the target underconditions favourable for binding, partitioning unbound nucleic acidsfrom those nucleic acids which have bound specifically to targetmolecules, dissociating the nucleic acid-target complexes, amplifyingthe nucleic acids dissociated from the nucleic acid-target complexes toyield a ligand-enriched library of nucleic acids, then reiterating thesteps of binding, partitioning, dissociating and amplifying through asmany cycles as desired to yield highly specific, high affinity nucleicacid ligands to the target molecule.

SELEX is based on the principle that within a nucleic acid librarycontaining a large number of possible sequences and structures there isa wide range of binding affinities for a given target. A nucleic acidlibrary comprising, for example a 20 nucleotide randomised segment canhave 4²⁰ structural possibilities. Those which have the higher affinityconstants for the target are considered to be most likely to bind. Theprocess of partitioning, dissociation and amplification generates asecond nucleic acid library, enriched for the higher binding affinitycandidates. Additional rounds of selection progressively favour the bestligands until the resulting library is predominantly composed of onlyone or a few sequences. These can then be cloned, sequenced andindividually tested for binding affinity as pure ligands.

Cycles of selection and amplification are repeated until a desired goalis achieved. In the most general case, selection/amplification iscontinued until no significant improvement in binding strength isachieved on repetition of the cycle. The iterativeselection/amplification method is sensitive enough to allow isolation ofa single sequence variant in a library containing at least 10¹⁴sequences. The method could, in principle, be used to sample as many asabout 10¹⁸ different nucleic acid species. The nucleic acids of thelibrary preferably include a randomised sequence portion as well asconserved sequences necessary for efficient amplification. Nucleic acidsequence variants can be produced in a number of ways includingsynthesis of randomised nucleic acid sequences and size selection fromrandomly cleaved cellular nucleic acids. The variable sequence portioncan contain fully or partially random sequence; it can also containsubportions of conserved sequence incorporated with randomised sequence.Sequence variation in test nucleic acids can be introduced or increasedby mutagenesis before or during the selection/amplification iterationsand by specific modification of cloned aptamers.

Antibodies

B-Raf polypeptides or peptides derived therefrom can be used to generateantibodies for use in the present invention. The B-Raf peptides usedpreferably comprise an epitope which is specific for a mutant B-Rafpolypeptide in accordance with the invention. Polypeptide fragmentswhich function as epitopes can be produced by any conventional means(see, for example, U.S. Pat. No. 4,631,211) In the present invention,antigenic epitopes preferably contain a sequence of at least 4, at least5, at least 6, at least 7, more preferably at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 20, at least 25, at least 30, at least 40, at least 50.and, most preferably, between about 15 to about 30 amino acids.Preferred polypeptides comprising immunogenic or antigenic epitopes areat least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or100 amino acid residues in length.

Antibodies can be generated using antigenic epitopes of B-Rafpolypeptides according to the invention by immunising animals, such asrabbits or mice, with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μg of peptide or carrier protein and Freund'sadjuvant or any other adjuvant known for stimulating an immune response.Several booster injections can be needed, for instance, at intervals ofabout two weeks, to provide a useful titre of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titre of anti-peptide antibodies inserum from an immunised animal can be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

The B-Raf polypeptides of the present invention, and immunogenic and/orantigenic epitope fragments thereof can be fused to other polypeptidesequences. For example, the polypeptides of the present invention can befused with immunoglobulin domains. Chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins have been shown to possess advantageous properties invivo (see, for example, EP 0394827; Traunecker et al., (1988) Nature,331: 84-86). Enhanced delivery of an antigen across the epithelialbarrier to the immune system has been demonstrated for antigens (such asinsulin) conjugated to an FcRn binding partner such as IgG or Fcfragments (see, for example, WO 96/22024 and WO 99/04813).

Moreover, the polypeptides of the present invention can be fused tomarker sequences, such as a peptide which facilitates purification ofthe fused polypeptide. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Another peptide tag useful for purification, the “HA”tag, corresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., (1984) Cell 37: 767. Thus, any of these abovefusions can be engineered using the nucleic acids or the polypeptides ofthe present invention.

In a preferred embodiment, the invention provides antibodies whichspecifically recognise B-Raf mutants as described herein.

Antibodies as described herein are especially indicated for diagnosticapplications. Accordingly, they can be altered antibodies comprising aneffector protein such as a label. Especially preferred are labels whichallow the imaging of the distribution of the antibody in vivo. Suchlabels can be radioactive labels or radioopaque labels, such as metalparticles, which are readily visualisable within the body of a patient.Moreover, they can be fluorescent labels or other labels which arevisualisable on tissue

Recombinant DNA technology can be used to improve the antibodies of theinvention. Thus, chimeric antibodies can be constructed in order todecrease the immunogenicity thereof in diagnostic or therapeuticapplications. Moreover, immunogenicity can be minimised by humanisingthe antibodies by CDR grafting [see European Patent Application 0 239400 (Winter)] and, optionally, framework modification [EP 0 239 400;Riechmann, L. et al., Nature, 332, 323-327, 1988; Verhoeyen M. et al.,Science, 239, 1534-1536, 1988; Kettleborough, C. A. et al., ProteinEngng., 4, 773-783, 1991; Maeda, H. et al., Human Antibodies andHybridoma, 2, 124-134, 1991; Gorman S. D. et al., Proc. Natl. Acad. Sci.USA, 88, 4181-4185, 1991; Tempest P. R. et al., Bio/Technology, 9,266-271, 1991; Co, M. S. et al., Proc. Natl. Acad. Sci. USA, 88,2869-2873, 1991; Carter, P. et al., Proc. Natl. Acad. Sci. USA, 89,4285-4289, 1992; Co, M. S. et al., J. Immunol., 148, 1149-1154, 1992;and, Sato, K. et al., Cancer Res., 53, 851-856, 1993].

Antibodies as described herein can be produced in cell culture.Recombinant DNA technology can be used to produce the antibodiesaccording to established procedure, in bacterial or preferably mammaliancell culture. The selected cell culture system optionally secretes theantibody product, although antibody products can be isolated fromnon-secreting cells.

Therefore, the present invention includes a process for the productionof an antibody according to the invention comprising culturing a host,e.g. E. coli, an insect cell or a mammalian cell, which has beentransformed with a hybrid vector comprising an expression cassettecomprising a promoter operably linked to a first DNA sequence encoding asignal peptide linked in the proper reading frame to a second DNAsequence encoding said antibody protein, and isolating said protein.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which are the customary standardculture media, for example Dulbecco's Modified Eagle Medium (DMEM) orRPMI 1640 medium, optionally replenished by a mammalian serum, e.g.foetal calf serum, or trace elements and growth sustaining supplements,e.g. feeder cells such as normal mouse peritoneal exudate cells, spleencells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin,low density lipoprotein, oleic acid, or the like. Multiplication of hostcells which are bacterial cells or yeast cells is likewise carried outin suitable culture media known in the art, for example for bacteria inmedium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, or M9Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, orComplete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up to give large amounts of the desired antibodies.Techniques for bacterial cell, yeast or mammalian cell cultivation areknown in the art and include homogeneous suspension culture, e.g. in anairlift reactor or in a continuous stirrer reactor, or immobilised orentrapped cell culture, e.g. in hollow fibres, microcapsules, on agarosemicrobeads or ceramic cartridges.

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumours. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristane, and, after one to two weeks,ascitic fluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, incorporated herein by reference. Techniques for the preparationof recombinant antibody molecules is described in the above referencesand also in, for example, EP 0623679; EP 0368684 and EP 0436597, whichare incorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies,preferentially by an enzyme immunoassay, e.g. a sandwich assay or adot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid can be concentrated, e.g. byprecipitation with ammonium sulphate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinitychromatography with the target antigen, or with Protein-A.

The invention further concerns hybridoma cells secreting the monoclonalantibodies of the invention. The preferred hybridoma cells of theinvention are genetically stable, secrete monoclonal antibodies of theinvention of the desired specificity and can be activated fromdeep-frozen cultures by thawing and recloning.

The invention, in a preferred embodiment, relates to the production ofanti mutant B-Raf antibodies. Thus, the invention also concerns aprocess for the preparation of a hybridoma cell line secretingmonoclonal antibodies according to the invention, characterised in thata suitable mammal, for example a Balb/c mouse, is immunised with a oneor more PDGF polypeptides or antigenic fragments thereof, or anantigenic carrier containing a mutant B-Raf polypeptide;antibody-producing cells of the immunised mammal are fused with cells ofa suitable myeloma cell line, the hybrid cells obtained in the fusionare cloned, and cell clones secreting the desired antibodies areselected. For example spleen cells of Balb/c mice immunised with mutantB-Raf are fused with cells of the myeloma cell line PAI or the myelomacell line Sp2/0-Ag14, the obtained hybrid cells are screened forsecretion of the desired antibodies, and positive hybridoma cells arecloned.

Preferred is a process for the preparation of a hybridoma cell line,characterised in that Balb/c mice are immunised by injectingsubcutaneously and/or intraperitoneally between 1 and 100 μg mutantB-Raf and a suitable adjuvant, such as Freund's adjuvant, several times,e.g. four to six times, over several months, e.g. between two and fourmonths, and spleen cells from the immunised mice are taken two to fourdays after the last injection and fused with cells of the myeloma cellline PAI in the presence of a fusion promoter, preferably polyethyleneglycol. Preferably the myeloma cells are fused with a three- totwentyfold excess of spleen cells from the immunised mice in a solutioncontaining about 30% to about 50% polyethylene glycol of a molecularweight around 4000. After the fusion the cells are expanded in suitableculture media as described hereinbefore, supplemented with a selectionmedium, for example HAT medium, at regular intervals in order to preventnormal myeloma cells from overgrowing the desired hybridoma cells.

The invention also concerns recombinant nucleic acids comprising aninsert coding for a heavy chain variable domain and/or for a light chainvariable domain of antibodies directed to mutant B-Raf as describedhereinbefore. By definition such DNAs comprise coding single strandedDNAs, double stranded DNAs consisting of said coding DNAs and ofcomplementary DNAs thereto, or these complementary (single stranded)DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or for alight chain variable domain of antibodies directed to mutant B-Raf canbe enzymatically or chemically synthesised DNA having the authentic DNAsequence coding for a heavy chain variable domain and/or for the lightchain variable domain, or a mutant thereof. A mutant of the authenticDNA is a DNA encoding a heavy chain variable domain and/or a light chainvariable domain of the above-mentioned antibodies in which one or moreamino acids are deleted or exchanged with one or more other amino acids.Preferably said modification(s) are outside the CDRs of the heavy chainvariable domain and/or of the light chain variable domain of theantibody. Such a mutant DNA is also intended to be a silent mutantwherein one or more nucleotides are replaced by other nucleotides withthe new codons coding for the same amino acid(s). Such a mutant sequenceis also a degenerated sequence. Degenerated sequences are degeneratedwithin the meaning of the genetic code in that an unlimited number ofnucleotides are replaced by other nucleotides without resulting in achange of the amino acid sequence originally encoded. Such degeneratedsequences can be useful due to their different restriction sites and/orfrequency of particular codons which are preferred by the specific host,particularly E. coli, to obtain an optimal expression of the heavy chainmurine variable domain and/or a light chain murine variable domain.

In this context, the term mutant is intended to include a DNA mutantobtained by in vitro mutagenesis of the authentic DNA according tomethods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

The invention therefore also concerns recombinant nucleic acidscomprising an insert coding for a heavy chain murine variable domain ofan anti mutant B-Raf antibody fused to a human constant domain γ, forexample γ1, γ2, γ3 or γ4, preferably γ1 or γ4. Likewise the inventionconcerns recombinant DNAs comprising an insert coding for a light chainmurine variable domain of an anti mutant B-Raf antibody directed tomutant B-Raf fused to a human constant domain κ or λ, preferably κ.

In another embodiment the invention pertains to recombinant DNAs codingfor a recombinant polypeptide wherein the heavy chain variable domainand the light chain variable domain are linked by way of a spacer group,optionally comprising a signal sequence facilitating the processing ofthe antibody in the host cell and/or a DNA coding for a peptidefacilitating the purification of the antibody and/or a cleavage siteand/or a peptide spacer and/or an effector molecule.

Antibodies and antibody fragments according to the invention are usefulin diagnosis. Accordingly, the invention provides a composition fordiagnosis comprising an antibody according to the invention.

In the case of a diagnostic composition, the antibody is preferablyprovided together with means for detecting the antibody, which can beenzymatic, fluorescent, radioisotopic or other means. The antibody andthe detection means can be provided for simultaneous, simultaneousseparate or sequential use, in a diagnostic kit intended for diagnosis.

The antibodies of the invention can be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA, sandwich immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays and protein A immunoassays. Such assaysare routine in the art (see, for example, Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below.

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), exposing the membraneto a primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, exposing the membrane toa secondary antibody (which recognises the primary antibody, e.g., anantihuman antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotitre plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognises the antibody of interest) conjugated to a detectable compoundcan be added to the well. Further, instead of coating the well with theantigen, the antibody can be coated to the well. In this case, a secondantibody conjugated to a detectable compound can be added following theaddition of the antigen of interest to the coated well.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labelled antigen (e.g., ³H or ¹²⁵I) withthe antibody of interest in the presence of increasing amounts ofunlabeled antigen, and the detection of the antibody bound to thelabelled antigen. The affinity of the antibody of interest for aparticular antigen and the binding off-rates can be determined from thedata by scatchard plot analysis. Competition with a second antibody canalso be determined using radioimmunoassays. In this case, the antigen isincubated with antibody of interest conjugated to a labelled compound(e.g., ³H or ¹²⁵I) in the presence of increasing amounts of an unlabeledsecond antibody.

Preparation of Mutant B-Raf Polypeptides

Mutant B-Raf polypeptides in accordance with the present invention canbe produced by any desired technique, including chemical synthesis,isolation from biological samples and expression of a nucleic acidencoding such a polypeptide. Nucleic acids, in their turn, can besynthesised or isolated from biological sources of mutant B-Raf.

The invention thus relates to vectors encoding a polypeptide accordingto the invention, or a fragment thereof. The vector can be, for example,a phage, plasmid, viral, or retroviral vector.

Nucleic acids according to the invention can be part of a vectorcontaining a selectable marker for propagation in a host. Generally, aplasmid vector is introduced in a precipitate, such as a calciumphosphate precipitate, or in a complex with a charged lipid. If thevector is a virus, it can be packaged in vitro using an appropriatepackaging cell line and then transduced into host cells.

The nucleic acid insert is operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs. Other suitable promoters are known to those skilledin the art. The expression constructs further contain sites fortranscription initiation, termination, and, in the transcribed region, aribosome binding site for translation. The coding portion of thetranscripts expressed by the constructs preferably includes atranslation initiating codon at the beginning and a termination codon(UAA, UGA or UAG) appropriately positioned at the end of the polypeptideto be translated.

As indicated, the expression vectors preferably include at least oneselectable marker. Such markers include dihydrofolate reductase, G418 orneomycin resistance for eukaryotic cell culture and tetracycline,kanamycin or ampicillin resistance genes for culturing in E. coli andother bacteria. Representative examples of appropriate hosts include,but are not limited to, bacterial cells, such as E. coli, Streptomycesand Salmonella typhimurium cells; fungal cells, such as yeast cells(e.g., Saccharomyces cerevisiae or Pichia pastoris); insect cells suchas Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO,COS, 293, and Bowes melanoma cells; and plant cells.

Appropriate culture media and conditions for the above-described hostcells are known in the art and available commercially.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK2233, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPA0815 (all available from Invitrogen, Carlsbad, Calif.).

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Sambrook et al., referred to above.

A polypeptide according to the invention can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulphate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides according to the present invention can also be recoveredfrom biological sources, including bodily fluids, tissues and cells,especially cells derived from tumour tissue or suspected tumour tissuesfrom a subject.

In addition, polypeptides according to the invention can be chemicallysynthesised using techniques known in the art (for example, seeCreighton, 1983, Proteins: Structures and Molecular Principles, W. H.Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310: 105-111(1984)). For example, a polypeptide corresponding to a fragment of amutant B-Raf polypeptide can be synthesised by use of a peptidesynthesiser.

B-Raf Mutations

Mutations in B-Raf have been identified in human tumour cells. Table 1describes the location of these mutations and the tumours in which theywere identified. The mutations are in the kinase domain of B-Raf. Mostof the mutations can be confirmed as somatic, indicating that a pairednormal/tumour sample was tested and the mutation found only in thetumour sample.

TABLE 1 cDNA accession Protein accession Nucleic acid Gene No. No.mutation Protein mutation Tumour Tumour type Somatic B-Raf NM_004333NP_004324 T1796A V599E A101D melanoma N/a B-Raf NM_004333 NP_004324T1796A V599E A2058 melanoma N/a B-Raf NM_004333 NP_004324 T1796A V599EA375 melanoma N/a B-Raf NM_004333 NP_004324 T1796A V599E A673 Sarcoma(Ewings) N/a B-Raf NM_004333 NP_004324 T1796A V599E C32 melanoma N/aB-Raf NM_004333 NP_004324 T1796A V599E COLO-205 colorectal N/a B-RafNM_004333 NP_004324 T1796A V599E COLO-679 melanoma N/a B-Raf NM_004333NP_004324 T1796A V599E COLO-741 colorectal N/a B-Raf NM_004333 NP_004324T1796A V599E COLO-800 melanoma N/a B-Raf NM_004333 NP_004324 T1796AV599E Colo829 Malignant Yes melanoma B-Raf NM_004333 NP_004324 T1796AV599E Colo-829 Melanoma cell line Yes pair p-loop Colon B-Raf NM_004333NP_004324 T1796A V599E DBTRG- glioma N/a 05MG B-Raf NM_004333 NP_004324T1796A V599E DU-4475 Breast cancer N/a B-Raf NM_004333 NP_004324 T1796AV599E DU-4475 Breast N/a B-Raf NM_004333 NP_004324 T1796A V599E Duke Mel103 melanoma Yes B-Raf NM_004333 NP_004324 T1796A V599E Duke Mel 104melanoma Yes B-Raf NM_004333 NP_004324 G1394C G465A Duke Mel 105melanoma Yes B-Raf NM_004333 NP_004324 T1796A V599E Duke Mel 108melanoma Yes B-Raf NM_004333 NP_004324 T1796A V599E Duke Mel 110melanoma Yes B-Raf NM_004333 NP_004324 T1796A V599E Duke Mel 111melanoma Yes B-Raf NM_004333 NP_004324 T1796A V599E Duke Mel 113melanoma Yes B-Raf NM_004333 NP_004324 T1796A V599E Duke Mel 115melanoma Yes B-Raf NM_004333 NP_004324 T1796A V599E Duke Mel 114melanoma Yes B-Raf NM_004333 NP_004324 T1796A V599E G-361 melanoma N/aB-Raf NM_004333 NP_004324 T1796A V599E GCT Sarcoma N/a(GCT/histiocytoma) B-Raf NM_004333 NP_004324 T1796A V599E HT-144melanoma N/a B-Raf NM_004333 NP_004324 T1796A V599E HT29 Colorectalcancer N/a B-Raf NM_004333 NP_004324 T1796A V599E HT29 colorectal N/aB-Raf NM_004333 NP_004324 G1388A G463E Hx62-26 Ovary/bladder? N/a B-RafNM_004333 NP_004324 T1796A V599E HxLL melanoma N/a B-Raf NM_004333NP_004324 T1796A V599E KG-1-C glioma N/a B-Raf NM_004333 NP_004324T1796A V599E LS-411N colorectal N/a p-loop Lung B-Raf NM_004333NP_004324 T1796A V599E Malme-3M Malignant N/a melanoma B-Raf NM_004333NP_004324 T1796A V599E Malme-3M melanoma N/a B-Raf NM_004333 NP_004324G1388T G463V MDA-MB- Breast N/a 231 B-Raf NM_004333 NP_004324 T1796AV599E MDA-MB- Breast cancer N/a 435 B-Raf NM_004333 NP_004324 T1796AV599E MDA-MB- Breast N/a 435 B-Raf NM_004333 NP_004324 G1403C G468ANCI-H1395 NSCLC N/a B-Raf NM_004333 NP_004324 G1394T G465V NCI-H1666NSCLC N/a B-Raf NM_004333 NP_004324 G1403C G468A NCI-H1755 NSCLC N/aB-Raf NM_004333 NP_004324 C1786G L596V NCI-H2087 NSCLC Yes B-RafNM_004333 NP_004324 C1786G L596V NCI-H2087 NSCLC N/a B-Raf NM_004333NP_004324 G1783C G595R NCI-H508 colorectal N/a B-Raf NM_004333 NP_004324T1796A V599E NMC-G1 glioma N/a B-Raf NM_004333 NP_004324 T1795A V599EOv-90-93 Ovarian cancer Yes B-Raf NM_004333 NP_004324 T1796A V599EPT-18-92-T Ovarian cancer Yes B-Raf NM_004333 NP_004324 G1753A E585KPT-52-91-T Ovarian cancer Yes B-Raf NM_004333 NP_004324 T1787G L596RPT-66-91-T Ovarian cancer Yes B-Raf NM_004333 NP_004324 T1796A V599EPT-93-13956-T Colon cancer, hk Yes B-Raf NM_004333 NP_004324 T1796AV599E PT-93-7014T Colon cancer, HK Yes B-Raf NM_004333 NP_004324 T1796AV599E PT-93-7014-T Colon cancer, HK Yes B-Raf NM_004333 NP_004324 T1782GF594L PT-97-51-T Colon Yes adenocarcinoma, Duke B-Raf NM_004333NP_004324 C1786G L596V PT-97-51-T Colon Yes adenocarcinoma, Duke B-RafNM_004333 NP_004324 T1796A V599E PT-97-93-T Ovarian cancer Yes B-RafNM_004333 NP_004324 T1796A V599E RPMI-7951 melanoma N/a B-Raf NM_004333NP_004324 T1796A V599E RUCH2-DH rhabdomyosarcoma N/a B-Raf NM_004333NP_004324 T1796A V599E Sarcoma 24 Histiocytoma Yes B-Raf NM_004333NP_004324 T1796A V599E SH-4 melanoma N/a B-Raf NM_004333 NP_004324T1796A V599E S86-5261 melanoma N/a B-Raf NM_004333 NP_004324 T1796AV599E S93-11360 melanoma N/a B-Raf NM_004333 NP_004324 T1796A V599ES94-6209 melanoma N/a B-Raf NM_004333 NP_004324 T1796A V599E S95-10334melanoma N/a B-Raf NM_004333 NP_004324 T1796A V599E S99-11631 melanomaN/a B-Raf NM_004333 NP_004324 T1796A V599E SK-HEP-1 hepatocellular N/aB-Raf NM_004333 NP_004324 T1796A V599E SK-MEL-24 melanoma N/a B-RafNM_004333 NP_004324 T1796A V599E SK-MEL-28 melanoma N/a B-Raf NM_004333NP_004324 T1796A V599E SK-MEL-3 melanoma N/a B-Raf NM_004333 NP_004324T1796A V599E SW1417 colorectal N/a B-Raf NM_004333 NP_004324 T1796AV599E SW872 Sarcoma N/a (liposarcoma) B-Raf NM_004333 NP_004324 T1796AV599E TE-159-T rhabdomyosarcoma N/a B-Raf NM_004333 NP_004324 T1796AV599E AM-38 glioma N/a B-Raf NM_004333 NP_004324 TG1796-97AT V599EWM-266- melanoma N/a 4/WM-115 B-Raf NM_004333 NP_004324 G2041A R681QHEC-1-A Uterus N/a B-Raf NM_004333 NP_004324 T974C I325T ZR-75-30 BreastN/a

Compound Assays

According to the present invention, mutant B-Raf is used as a target toidentify compounds, for example lead compounds for pharmaceuticals,which are capable of modulating the proliferative activity of mutantB-Raf. Accordingly, the invention relates to an assay and provides amethod for identifying a compound or compounds capable, directly orindirectly, of modulating the activity mutant B-Raf, comprising thesteps of:

-   -   (a) incubating mutant B-Raf with the compound or compounds to be        assessed; and    -   (b) identifying those compounds which influence the activity of        mutant B-Raf.

Mutant B-Raf is as defined in the context of the present invention.

According to a first embodiment of this aspect invention, the assay isconfigured to detect polypeptides which bind directly to mutant B-Raf.

The invention therefore provides a method for identifying a modulatorcell proliferation, comprising the steps of

-   -   (a) incubating mutant B-Raf with the compound or compounds to be        assessed; and    -   (b) identifying those compounds which bind to mutant B-Raf.

Preferably, the method further comprises the step of:

-   -   (c) assessing the compounds which bind to mutant B-Raf for the        ability to modulate cell proliferation in a cell-based assay.

Binding to mutant B-Raf may be assessed by any technique known to thoseskilled in the art. Examples of suitable assays include the two hybridassay system, which measures interactions in vivo, affinitychromatography assays, for example involving binding to polypeptidesimmobilised on a column, fluorescence assays in which binding of thecompound(s) and mutant B-Raf is associated with a change in fluorescenceof one or both partners in a binding pair, and the like. Preferred areassays performed in vivo in cells, such as the two-hybrid assay.

In a preferred aspect of this embodiment, the invention provides amethod for identifying a lead compound for a pharmaceutical useful inthe treatment of disease involving or using cell proliferation,comprising incubating a compound or compounds to be tested with mutantB-Raf, under conditions in which, but for the presence of the compoundor compounds to be tested, B-Raf associates with RAS with a referenceaffinity;

-   -   determining the binding affinity of mutant B-Raf for RAS in the        presence of the compound or compounds to be tested; and    -   selecting those compounds which modulate the binding affinity of        mutant B-Raf for RAS with respect to the reference binding        affinity.

Preferably, therefore, the assay according to the invention iscalibrated in absence of the compound or compounds to be tested, or inthe presence of a reference compound whose activity in binding to mutantB-Raf is known or is otherwise desirable as a reference value. Forexample, in a two-hybrid system, a reference value may be obtained inthe absence of any compound. Addition of a compound or compounds whichincrease the binding affinity of mutant B-Raf for a target increases thereadout from the assay above the reference level, whilst addition of acompound or compounds which decrease this affinity results in a decreaseof the assay readout below the reference level.

In a second embodiment, the invention may be configured to detectfunctional interactions between a compound or compounds and mutantB-Raf. Such interactions will occur either at the level of theregulation of mutant B-Raf, such that this kinase is itself activated orinactivated, for example by RAS, in response to the compound orcompounds to be tested, or at the level of the modulation of thebiological effect of mutant B-Raf on downstream targets such as MEK. Asused herein, “activation” and “inactivation” include modulation of theactivity, enzymatic or otherwise, of a compound, as well as themodulation of the rate of production thereof, for example by theactivation or repression of expression of a polypeptide in a cell. Theterms include direct action on gene transcription in order to modulatethe expression of a gene product.

Assays which detect modulation of the functional interaction betweenmutant B-Raf and its upstream or downstream partners in a signallingpathway are preferably cell-based assays. For example, they may be basedon an assessment of the degree of phosphorylation of MAPK, which isindicative of the degree of MEK activation, resulting from activation ofmutant B-Raft.

In preferred embodiments, a nucleic acid encoding mutant B-Raf isligated into a vector, and introduced into suitable host cells toproduce transformed cell lines that express mutant B-Raft. The resultingcell lines can then be produced for reproducible qualitative and/orquantitative analysis of the effect(s) of potential compounds affectingmutant B-Raf function. Thus mutant B-Raf expressing cells may beemployed for the identification of compounds, particularly low molecularweight compounds, which modulate the function of mutant B-Raf. Thus hostcells expressing mutant B-Raf are useful for drug screening and it is afurther object of the present invention to provide a method foridentifying compounds which modulate the activity of mutant B-Raf, saidmethod comprising exposing cells containing heterologous DNA encodingmutant B-Raf, wherein said cells produce functional mutant B-Raf, to atleast one compound or mixture of compounds or signal whose ability tomodulate the activity of said mutant B-Raf is sought to be determined,and thereafter monitoring said cells for changes caused by saidmodulation. Such an assay enables the identification of modulators, suchas agonists, antagonists and allosteric modulators, of mutant B-Raf. Asused herein, a compound or signal that modulates the activity of mutantB-Raf refers to a compound that alters the activity of mutant B-Raf insuch a way that the activity of mutant B-Raf on a target thereof, suchas MEK, is different in the presence of the compound or signal (ascompared to the absence of said compound or signal).

Cell-based screening assays can be designed by constructing cell linesin which the expression of a reporter protein, i.e. an easily assayableprotein, such as β-galactosidase, chloramphenicol acetyltransferase(CAT) or luciferase, is dependent on the activation of a mutant B-Rafsubstrate. For example, a reporter gene encoding one of the abovepolypeptides may be placed under the control of an response elementwhich is specifically activated by MEK or MAPK. Such an assay enablesthe detection of compounds that directly modulate mutant B-Raf function,such as compounds that antagonise phosphorylation of MEK by mutantB-Raf, or compounds that inhibit or potentiate other cellular functionsrequired for the activity of mutant B-Raf. Cells in which wild-type,non-mutant B-Raf is present provide suitable controls.

Alternative assay formats include assays which directly assessproliferative responses in a biological system. The constitutiveexpression of unregulated mutant B-Raf results in an proliferativephenotype in animal cells. Cell-based systems, such as 3T3 fibroblasts,may be used to assess the activity of potential regulators of mutantB-Raf.

In a preferred aspect of this embodiment of the invention, there isprovided a method for identifying a lead compound for a pharmaceuticaluseful in the treatment of disease involving or using an inflammatoryresponse, comprising:

-   -   incubating a compound or compounds to be tested with mutant        B-Raf and MEK, under conditions in which, but for the presence        of the compound or compounds to be tested, mutant B-Raf directly        or indirectly causes the phosphorylation of MEK with a reference        phosphorylation efficiency;    -   determining the ability of mutant B-Raf to cause the        phosphorylation, directly or indirectly, of MEK in the presence        of the compound or compounds to be tested; and    -   selecting those compounds which modulate the ability of mutant        B-Raf to phosphorylate MEK with respect to the reference        phosphorylation efficiency.

In a further preferred aspect, the invention relates to a method foridentifying a lead compound for a pharmaceutical, comprising the stepsof:

-   -   providing a purified mutant B-Raf molecule;    -   incubating the mutant B-Raf molecule with a substrate known to        be phosphorylated by mutant B-Raf and a test compound or        compounds; and    -   identifying the test compound or compounds capable of modulating        the phosphorylation of the substrate.

A substrate for mutant B-Raf phosphorylation is MEK. Preferably,therefore, MEK is used as a substrate to monitor compounds capable ofmodulating mutant B-Raf kinase activity. This allows the person skilledin the art to screen directly for kinase modulators. Preferably, kinasemodulators are kinase (mutant B-Raf) inhibitors.

In a preferred embodiment, the activity of 13-Raf may be measuredaccording to the following protocol:

-   -   1. Cells are solubilized in lysis buffer (150 mM NaCl, 25 mM        HEPES [pH 7.3], 1 mM sodium orthovanadate, 1% Triton X-100,        protease inhibitors, 0.5 mM dithiothreitol).    -   2. The lysate is incubated on ice for 10 min and centrifuged at        14,000 3 g for 10 min, and the supernatant incubated with        polyclonal anti-B-Raf antibody and then with protein G-Sepharose        at 4° C. for 1 h.    -   1. The immunoprecipitates are washed twice with lysis buffer,        and the kinase reaction carried out at 30° C. for 10 min in        kinase buffer (0.2 mM ATP, 30 mM MgCl₂, 2 mM MnCl₂, 40 mM sodium        β-glycerophosphate, 0.2 mM sodium orthovanadate, 2 mM okadaic        acid, 0.2% β-mercaptoethanol) with 1 mg of purified recombinant        MEK1 added as the substrate.    -   4. After MEK1 activation, 15 mCi of ^([γ-32P])ATP and 1 mg of        kinase-defective (K52R) Erk are added as the substrate for an        additional 2 min. The reaction is terminated by the addition of        sample buffer, the mixture was boiled for 5 min, and the        proteins separated by SDS-PAGE.    -   5. The gel proteins are transferred to a polyvinylidene        difluoride membrane, on which the amount of radiolabeled K52R        Erk is quantitated by a PhosphorImager.    -   6. For the calculations of B-Raf activity, the amount of B-Raf        protein on the same membrane is determined by probing the        membrane with ¹²⁵I-labeled goat anti-mouse IgG following mouse        monoclonal anti-B-Raf blotting.    -   7. The assay can be repeated in the presence or absence of        compound(s) to be tested.

Optionally, the test compound(s) identified may then be subjected to invivo testing to determine their effects on a mutant B-Raf signallingpathway, for example as set forth in the foregoing embodiment.

As used herein, “mutant B-Raf activity” may refer to any activity ofmutant B-Raf, including its binding activity, but in particular refersto the phosphorylating activity of mutant B-Raf. Accordingly, theinvention may be configured to detect the phosphorylation of targetcompounds by mutant B-Raf, and the modulation of this activity bypotential therapeutic agents.

Examples of compounds which modulate the phosphorylating activity ofmutant B-Raf include dominant negative mutants of B-Raf itself. Suchcompounds are able to compete for the target of mutant B-Raf, thusreducing the activity of mutant B-Raf in a biological or artificialsystem. Thus, the invention moreover relates to compounds capable ofmodulating the phosphorylating activity of mutant B-Raf.

Compounds which influence the activity of mutant B-Raf may be of almostany general description, including low molecular weight compounds,including organic compounds which may be linear, cyclic, polycyclic or acombination thereof, peptides, polypeptides including antibodies, orproteins. In general, as used herein, “peptides”, “polypeptides” and“proteins” are considered equivalent.

Many compounds according to the present invention may be lead compoundsuseful for drug development. Useful lead compounds are especiallyantibodies and peptides, and particularly intracellular antibodiesexpressed within the cell in a gene therapy context, which may be usedas models for the development of peptide or low molecular weighttherapeutics. In a preferred aspect of the invention, lead compounds andmutant B-Raf or other target peptides may be co-crystallised in order tofacilitate the design of suitable low molecular weight compounds whichmimic the interaction observed with the lead compound.

Crystallisation involves the preparation of a crystallisation buffer,for example by mixing a solution of the peptide or peptide complex witha “reservoir buffer”, preferably in a 1:1 ratio, with a lowerconcentration of the precipitating agent necessary for crystalformation. For crystal formation, the concentration of the precipitatingagent is increased, for example by addition of precipitating agent, forexample by titration, or by allowing the concentration of precipitatingagent to balance by diffusion between the crystallisation buffer and areservoir buffer. Under suitable conditions such diffusion ofprecipitating agent occurs along the gradient of precipitating agent,for example from the reservoir buffer having a higher concentration ofprecipitating agent into the crystallisation buffer having a lowerconcentration of precipitating agent. Diffusion may be achieved forexample by vapour diffusion techniques allowing diffusion in the commongas phase. Known techniques are, for example, vapour diffusion methods,such as the “hanging drop” or the “sitting drop” method. In the vapourdiffusion method a drop of crystallisation buffer containing the proteinis hanging above or sitting beside a much larger pool of reservoirbuffer. Alternatively, the balancing of the precipitating agent can beachieved through a semipermeable membrane that separates thecrystallisation buffer from the reservoir buffer and prevents dilutionof the protein into the reservoir buffer.

In the crystallisation buffer the peptide or peptide/binding partnercomplex preferably has a concentration of up to 30 mg/ml, preferablyfrom about 2 mg/ml to about 4 mg/ml.

Formation of crystals can be achieved under various conditions which areessentially determined by the following parameters: pH, presence ofsalts and additives, precipitating agent, protein concentration andtemperature. The pH may range from about 4.0 to 9.0. The concentrationand type of buffer is rather unimportant, and therefore variable, e.g.in dependence with the desired pH. Suitable buffer systems includephosphate, acetate, citrate, Tris, MES and HEPES buffers. Useful saltsand additives include e.g. chlorides, sulphates and other salts known tothose skilled in the art. The buffer contains a precipitating agentselected from the group consisting of a water miscible organic solvent,preferably polyethylene glycol having a molecular weight of between 100and 20000, preferentially between 4000 and 10000, or a suitable salt,such as a sulphates, particularly ammonium sulphate, a chloride, acitrate or a tartarate.

A crystal of a peptide or peptide/binding partner complex according tothe invention may be chemically modified, e.g. by heavy atomderivatization. Briefly, such derivatization is achievable by soaking acrystal in a solution containing heavy metal atom salts, or aorganometallic compounds, e.g. lead chloride, gold thiomalate,thimerosal or uranyl acetate, which is capable of diffusing through thecrystal and binding to the surface of the protein. The location(s) ofthe bound heavy metal atom(s) can be determined by X-ray diffractionanalysis of the soaked crystal, which information may be used e.g. toconstruct a three-dimensional model of the peptide.

A three-dimensional model is obtainable, for example, from a heavy atomderivative of a crystal and/or from all or part of the structural dataprovided by the crystallisation.

Preferably building of such model involves homology modelling and/ormolecular replacement.

The preliminary homology model can be created by a combination ofsequence alignment with any RAF kinase the structure of which is known,secondary structure prediction and screening of structural libraries.For example, the sequences of mutant B-Raf and a candidate peptide canbe aligned using a suitable software program.

Computational software may also be used to predict the secondarystructure of the peptide or peptide complex. The peptide sequence may beincorporated into the mutant B-Raf structure. Structural incoherences,e.g. structural fragments around insertions/deletions can be modelled byscreening a structural library for peptides of the desired length andwith a suitable conformation. For prediction of the side chainconformation, a side chain rotamer library may be employed.

The final homology model is used to solve the crystal structure of thepeptide by molecular replacement using suitable computer software. Thehomology model is positioned according to the results of molecularreplacement, and subjected to further refinement comprising moleculardynamics calculations and modelling of the inhibitor used forcrystallisation into the electron density.

Kinase Activation Studies

Constitutively active kinase mutants are valuable research tools in theelucidation of signalling pathways and the development of therapeuticagents which modulate such pathways. The activity of five of the mutantsaccording to the invention has been examined. These are G463V, G468A,0595R, L596V and V599E. To examine the activity of the mutants,myc-epitope tagged versions of B-Raf are transiently expressed in COScells. To examine the activity of this exogenously expressed B-Raf, theprotein is immunoprecipitated using the myc-tag and examined in a kinasecascade assay, using bacterially produced GST-MEK, GST-ERK and myelinbasic protein (MBP) as sequential substrates (Marais et al (1997); J.Biol. Chem. 272: 4378-83). B-Raf has high levels of basal kinaseactivity, being significantly more active in the absence of activatorsthan either Raf-1 or A-Raf (Marais et al (1997); J. Biol. Chem. 272:4378-83). Moreover, whereas Raf-1 and A-Raf require both oncogenic Ras(^(V12)Ras) and activated Src to stimulate their activity fully, B-Rafis fully activated by co-expression with ^(V12)Ras alone. The effectthese mutations have on both the basal activity of B-Raf and on theactivity stimulated by ^(V12)Ras is therefore assayed. Compared towild-type B-Raf, ^(G463V)B-Raf, ^(G468A)B-Raf, ^(L596V)B-Raf and^(V599E)B-Raf all possess strongly elevated basal kinase activity (FIG.1A, 1B). By comparison, ^(G595R)B-Raf has reduced basal activitycompared to the wild-type protein (FIG. 1A). Similar results areobserved in vivo. All five mutants are stimulated by oncogenic Ras(^(V12)Ras). However, the fold activation for each of the mutants isreduced compared with wild-type B-Raf (See FIG. 1A, B) and isparticularly small in the case of ^(V599E)B-Raf. However, since thebasal activity of each of ^(G463V)B-Raf, ^(G468A)B-Raf, ^(L596V)B-Rafand ^(V599E)B-Raf is higher than the wild-type protein, then absolutelevels of activity seen are higher in each case in the presence of^(V12)Ras than for the wild-type protein. Interestingly, ^(G595R)B-Rafis also stimulated by ^(V12)Ras, but the activation was very weak,probably due to the low starting levels.

The ability of each of the activated mutants to transform NIH3T3 cellsis also examined. In this assay, wild-type B-Raf transforms cells atvery low efficiency (˜0.02% of the number of colonies seen with^(V12)Ras). However, as shown in FIG. 2, the each of the activatedmutants transforms NIH3T3 cells 40 to 85 fold more efficiently than doeswild-type B-Raf.

The extent of dependence of the growth of cells that contain the B-Rafmutants on the Ras/MEK pathway is investigated. For these studies, twoassays are used. The =first is to test whether their growth issuppressed by micro-injection of the monoclonal antibody Y13-259, anantibody that neutralises the activity of cellular Ras. The results areshown in Table 2. The data are divided into three groups. The firstgroup have wild-type B-Raf and are their growth is inhibited (40-100%)by Y13-259. The second group have activating mutations in B-Raf andtheir growth was not inhibited (<15%) by Y13-259. The third group (onlyone case) contains a cell which has both an activating Ras and anactivating B-Raf mutation. Intriguingly, the growth of this cell linewas inhibited by Y13-259, but this may be because the growth isdependent on both Ras and B-Raf.

The second approach is to examine the effects of the compound U0126, aninhibitor of MEK1/2, the only known substrates for B-Raf. These resultsdemonstrate that treatment of cells that have activating mutations inB-Raf results in suppression of cell proliferation when MEK activity issuppressed indicating that the activation of cell signalling byactivated mutants of B-Raf is a therapeutic target. See Table 3.

Taken together, the above data demonstrate that

1. There are two classes of B-Raf mutation in human tumours, activatingand inactivating mutations.2. The activated versions of B-Raf are able to transform NIH3T3 cellsand so are can be defined as oncogenes.3. Human tumour cell lines that express activated B-Raf protein are notsensitive to Y13-259, a Ras neutralising antibody, indicating that theirgrowth is not dependent on Ras proteins and so are unlikely to respondto compounds that target the Ras proteins. This indicates that theactivating mutants may overcome the requirement for Ras signals intumour cells.4. However, their activity is suppressed by the compound U0126,indicating that their growth is dependent on the activity of thispathway and therefore likely to respond to therapeutic agents thattarget B-Raf activity.5. Since some of the mutations are in the phosphate-binding loop of thekinase domain (G463, G465, G468) and these amino acids are conserved inall kinases, these mutations represent a global and convenient mechanismto activate kinases. This has important implications in the screeningfor therapeutic agents.

The invention accordingly provides a constitutively active kinasecomprising a mutation in the phosphate binding loop thereof selectedfrom the group consisting of mutations at one or more positionscorresponding to positions 463, 465 and 468 of B-Raf.

Preferably, the mutation is at one or more of positions 463 and 468.

Most preferred are G463V and G468A.

Many kinases are identified to be associated with a specific disease,but their mechanisms of activation may not always be fully understood.Constitutively activated mutants thereof as described herein provide areagent that can be used to screen for inhibitors without having tofirst elucidate their mechanism of activation.

Exemplary kinases include other kinases on the MAP kinase pathway, suchas MEK and ERK and the other MAP kinase pathways, such as p38, JNK andtheir upstream kinases. Although something is known about theiractivation mechanisms, for some it is not known how to activate them bydirect mutation. The present invention provides activated mutants ofsaid kinases screening purposes. Moreover, kinases that are downstreamof the MAP kinases, such as p90Rsk, mnk, etc., can also be activated.Although alternative activation mechanisms are known, mutation may be apreferable route in screening assays.

The invention also encompasses certain known kinases which have no knownactivation mechanism, such as Lkb1, which is involved in Putz-jegerssyndrome; and kinase PDK1 which may be constitutively active, but whichcan be further activatable for drug screening. This kinase is involvedin insulin signalling, so may be a useful target for diabetes. Alsoinvolved in type II diabetes is the AMP-activated kinase, which again isactivated by phosphorylation and is therefore amenable to activation bymutation.

Therefore, the invention provides a method for screening one or morecompounds for an inhibitory effect on a kinase, comprising

-   -   (a) preparing a mutant kinase comprising an amino acid        substitution, deletion or insertion at one or more of positions        463, 465 or 468 as detailed above;    -   (b) exposing the mutant kinase to said one or more compounds in        the presence of a kinase substrate; and    -   (d) determining the ability of the kinase to phosphorylate the        substrate in the presence of the one or more compounds.

The phosphorylating activity of the kinase in the presence of the testcompound(s) is advantageously compared to its activity in the absence ofthe compound(s); a reduction in the basal activity of the mutant kinaseis indicative of inhibition of the kinase by the compound(s). Formultiple assays, a reference level of phosphorylation may be determinedfor a particular assay, and used as a basis for comparison.

Preferably, the kinase is a Raf protein kinase; advantageously, it isB-Raf.

Conversely, constitutively repressed mutants such as B-Raf G595R areuseful in screening for activators of a kinase.

Validation of bRAF as a Drug Target.

In order to validate BRAF as a target in cancer, it is first testedwhether the growth of cells that express activated, mutant forms of BRAFrequired the RAF-MEK-ERK signaling pathway for growth. To this end,melanoma and colorectal cell lines that harbour mutations in the BRAFgene are treated with pharmacological agents that block signalingthrough this pathway. Two compounds are tested. One is the compoundU0126, which is a MEK inhibitor and which therefore uncouples RAF-ERKsignaling in cells (Sebolt-Leopold et al., 1999). BAY 43-9006 os alsotested. This is an inhibitor of RAF proteins (Lyons et al., 2001). Theability of these compounds to block ERK activity was tested in themelanoma cell line WM266.4, which have substitution of an aspartic acidfor valine at position 599 of the BRAF gene. This is an activatingmutation (FIG. 3A). These cells also have elevated basal kinase activityas judged using an antibody (ppERK) that only binds to the doublyphosphorylated, activated version of ERK. When the ppERK antibody isused to Western blot WM266.4 cells, a strong signal is seen in theregion of 42-44 kDa, indicating that ERK has elevated basal kinaseactivity in these cells (FIG. 3B). However, when the cells are treatedwith U0126, or Bay 43-9006, ERK activity is strongly suppressed (FIG.3B). Similar results were obtained using A375 cell, a melanoma cell linethat harbours a V599E mutation in the BRAF gene (Davies et al., 2002).These data demonstrate that that RAF and MEK signaling is required forthe maintenance of the elevated ERK activity in these cells.

We next tested what effect BAY 43-9002 had on the growth of WM266.4cells and found that this compound blocked the growth of these cellswith an IC50 of ˜6.1 μM (Table 4). BAY 43-9006 also blocked the growthof colo 829 cells and of BE cells in the low micro-molar range (Table4). Colo 829 cells are a melanoma cell line that harbours a V599Emutation in the BRAF gene and BE cells are a colorectal line thatharbour a G463 mutation in the BRAF gene (Davies et al., 2002). As wehave shown, both of these mutations are activating (Davies et al.,2002). Finally, we tested the effects of these inhibitors on DNAsynthesis. Incubation of WM-266.4 cells with 10 μM U0126 or 10 μM BAY43-9006 strongly suppressed DNA synthesis in these cells (FIG. 4). Thesedata demonstrate that ERK activation and proliferation in cells thatharbour activating mutations in the BRAF gene are dependent on RAF andMEK activities.

There are three RAF genes in mammalian cells, CRAF (also called RAF-1),ARAF and BRAF. U0126 is a MEK inhibitor and therefore will not be ableto distinguish CRAF from BRAF or ARAF signaling. Similarly, BAY 43-9006can inhibit both CRAF and BRAF, so will not distinguish between thedifferent RAF isoforms. Therefore, in order to determine which RAFisoform was signaling to ERK in WM266.4, cells were treated with smallinterference RNA (siRNA) probes that are selective for the individualRAF isoforms. WM266.4 cells were treated with siRNA probes designed tobe specific for BRAF, or CRAF, or a scrambled control that should notrecognize either isoform. The efficiency of the recognition for CRAF wasdetermined by Western blotting. Treatment of WM266.4 cells with a siRNAprobe specific for CRAF resulted in strong suppression of CRAFexpression (FIG. 5A). Similar results were observed in Colo 829 cellsand BE cells (FIG. 5A). When WM-266.4 cells were treated with a BRAFspecific siRNA probe, BRAF activity in the cells was stronglysuppressed, but no suppression was observed when the cells were treatedwith the scrambled control (FIG. 5B). Similar results were observed inColo 829 and BE cells (FIG. 5B).

The above data show that siRNA can be used to selectively suppressexpression of the BRAF and CRAF proteins. We therefore examined howablation of each of these proteins affected ERK activity in these cells.When siRNA was used to ablate BRAF protein expression in WM-266.4, ERKactivity was suppressed in a time-dependent manner (FIG. 6). Bycontrast, ablation of CRAF expression or treatment with the scrambledsiRNA probes did not affect ERK activity (FIG. 6). Similar results wereobtained in Colo 829 cells (FIG. 6). These results demonstrate that BRAFis the major isoform that signals to ERK in melanoma cells that expressactivated BRAF proteins. CRAF does not appear to signal to basal ERKactivity in these cells.

Finally, we examined how BRAF ablation affected cell growth, examiningthe effects on apoptosis in WM-266.4 cells. For these studies, the cellswere fixed in 70% ethanol, stained with propridium iodide and their cellcycle profiles were examined by fluorescent activated cell sorting(FACS). Using this analysis, the apoptotic cells appear in the sub-G1peak. In these cells, spontaneous apoptosis is very low, with less than1% of the cell

Reagent Validation

appearing in the sub-G1 peak (FIG. 7, table 5). When the cells aretreated with U0126, the proportion of cells in the sub-G1 peak issignificantly increased (˜3.5%; FIG. 7, Table 5). Similarly, ablation ofBRAF expression by use of siRNA also increases the number of cells inthe sub-G1 peak, whereas ablation of CRAF or treatment with thescrambled control did not. We also examined PARP cleavage, a marker ofthe induction of apoptosis. Treatment of the cells with BRAF siRNAinduced cleavage of PARP, whereas the scrambled control did not. Thesedata demonstrate that when mutant BRAF protein is ablated in melanomacell lines, apoptosis is induced.

In summary, these results demonstrate that in melanoma cell lines thatexpress activated mutants of BRAF, signaling through RAF and MEK isrequired for ERK activation and for cell growth. BRAF, rather than CRAFappears to be the major RAF isoform that stimulates ERK activity, andappears to protect the cells from apoptosis. These data suggests thatBRAF is an important therapeutic target in cells that rely on BRAFsignaling for growth and protection from apoptosis.

Development of High-Throughput Screening Assay

A HTS assay has been developed for the B-raf mutant V599E. In order tovalidate results, -Raf-expressing lysate and GST-MKK1 reagents wereactivity checked by conducting a standard coupled assay employingGST-ERK2 (kinase competent) and measuring ³³P-γ-phosphate incorporationinto myelin basic protein (MBP). As shown in FIG. 8, in the presence ofthe B-Raf lysate a 16-fold increase in signal was observed compared tothe control (non-expressing) lysate.

Assay Platform Validation Option 1: Coupled Kinase Assay in GlutathioneFlashPlate

-   -   Principle of Platform: GST-tagged substrate (ERK-2) is captured        onto the scintillant-embedded walls of a flashplate via a        glutathione coating. The incorporation of ³³P-γ-phosphate into        substrate should result in a measurable scintillation signal.

The possibility of measuring the incorporation of ³³P-γ-phosphate intoGST-kinase dead ERK2 (GST-kdERK2) as an output of B-Raf activity wasevaluated in a glutathione flashplate assay. FIG. 9 demonstrates that,using the conditions transferred from the reagent validation exercise,we were unable to detect the B-Raf-dependent incorporation of ³³P intoGST-kdERK2 using this platform. A standard p81 filter plate assay alsoproved unsuccessful. As a consequence of the amplificationcharacteristics of this cascade assay, maintaining an assay signal inthe absence of the final assay step (ie. ERK2 phosphorylation of MBP)would most likely require significantly increased levels of each theremaining constituents. It was therefore deemed appropriate to assessthe antibody-based platform prior to embarking upon the reagent-costlyexercise of B-Raf, MKKI and ERK2 titrations in this radiometricplatform.

Option 2: Coupled Kinase Assay in DELFIA Format

DELFIA (Dissociation-Enhanced Lanthanide Fluorescence ImmunoAssay) assayinvolves the measurement of ERK2 phosphorylation via binding of aphospho-specific antibody. The coupled kinase assayB-Raf/GST-MKKI/GST-kdERK2 generates phosphorylated GST-kdERK2. Ananti-GST-coated plate is used to capture the GST-kdERK2. A primaryantibody is added that specifically detects ERK2 phosphorylated onThreonine and Tyrosine. A Europium {Eu)-labelled secondary antibody isthen added. In the presence of Enhancement Solution, the Eu-labeldissociates from the antibody absorbing at 335 nm and allowingfluorometric detection at an emission wavelength of 620 nm.

The B-Raf assay was assessed in this platform employing a combination ofkinase assay conditions from the reagent validation exercise andstandard DELFIA assay conditions. FIG. 10 shows that in the presence ofthe B-Raf lysate a 12.2-fold increase in signal was observed compared tothe control (non-expressing) lysate. The signal observed was entirelydependent upon the presence of all three enzyme/substrate components.

Based on preliminary experiments, the DELFIA platform was selected fordevelopment.

Kinase Assay Development B-Raf Lysate

Three batches of B-Raf lysate have been used throughout the procedure.Batch A was employed to establish the DELFIA assay platform. Batch B hasbeen used during assay development. For Batches B and C an approximatelinear relationship between lysate quantity and level of signal attainedwas evident between 0.025 to 0.1 μl per well. The final quantitiesselected for each batch were based upon attaining a sufficient window ofsignal within the linear range. As a result of these assessments, BatchA was used at 1 μl/well [96-well] and Batches B and C (screening) havebeen used at 0.1 μl/well [96-well] and 0.05 μl/well [384-well].

Optimisation of Antibody Levels

-   -   Initial concerns regarding the possible competition of ERK2 and        MKKI (both GST-tagged) for glutathione binding sites resulted in        early development assays being conducted using a ‘pre-binding        protocol’.        These Conditions were as Follows:    -   Pre-binding of 100 ng/well GST-kdERK2 to 96-well        Glutathione-coated plates.    -   Addition of B-Raf lysate (Batch A), MKK1 (6.5 μg/ml) and ATP 500        μM) in a final volume of 50 μM DKB (see Appendix I) and        incubation at 30° C. for 1 hour.

This protocol was employed to optimise and economise the antibody loadof the detection system. Titrations of both primary and secondaryantibodies were conducted to assess the possibility of reducing antibodylevels whilst maintaining a signal to background ratio of ≧10.1. FIGS. 4and 5 indicate that 1:3000 and 1:1000 were the lowest concentrationsacceptable for phosphoERK2 and Eu-Labelled antibodies respectively. Allsubsequent assays were therefore performed using these antibodyconcentrations.

Optimisation of MKK1 and ERK2 Levels

The ability to perform the enzyme component of this assay ‘in solution’without a pre-binding step was investigated to enable titration ofdefined concentrations of both MKK1 and ERK2. In addition, the reductionof this assay into a single-step (homogeneous) mixing of reagentsinvolved in the kinase would make it more amenable to HTS. FIG. 13illustrates that the homogenous assay and the pre-binding protocol gaveequivalent data. All subsequent assays were performed using the‘homogeneous’ protocol.

Titration of ERK-2 whilst maintaining MKK1 levels indicated that maximumsignal was attained at a ratio of ˜12:1 (ERK2:MKK1). In an attempt toeconomise on reagents a matrix titration of these components wasperformed. Using 0.1 μl B-Raf (Batch B), the combination of 6. 5 μg/mlGST-MKK1 and 80 μg/ml GST-kdERK2 gave the maximal signal and althoughsome reduction in the Raf/MKK1 load was possible it was deemedappropriate to maintain the original ratio. This decision was taken withthe knowledge that further assay parameter alterations (e.g. transfer to384; reduction of ATP load, transfer to automation) may reduce thesignal window further. All subsequent assays were, therefore, performedusing 6.5 μg/ml GST-MKK1 and 80 μg/ml GST-kdFRK2.

Effect of Temperature

-   -   The ability to run this assay at room temperature would        significantly simplify the eventual HTS process. The assay        signal at room temperature and 30° C. was therefore        investigated. Based upon the results obtained, the        signal-to-background ratio was acceptable at room temperature        and all subsequent assays were carried out at room temperature.

Evaluation of Assay in 384-Well Plate Format

-   -   In an attempt to enhance throughput and minimise reagent usage        during the screen, the performance of the assay in 384-well        format was assessed. In this format the assay performed well and        both standard (final of 50 μl) and reduced reagent volume (25        μl) assays returned highly acceptable signal to background        ratios. All subsequent development experiments were performed in        384-format using 25 μl reaction volumes.

Optimisation of ATP Concentration

-   -   The ATP concentration of a kinase screening assay has the        potential to influence the number and nature of inhibitory        compounds identified. Definition of the ATP levels of such an        assay is a balance of the following considerations:    -   employing ATP levels that enable a consistent, measurable window        of assay signal    -   employing ATP levels low enough to permit the identification of        ATP-competitive inhibitors    -   employing ATP levels sufficiently high such that weak        ATP-competitive agents likely to be ineffective in the context        of cellular ATP are less likely to be detected

Kinase screening assays are usually performed at an ATP concentrationrelative to Km. The derivation of Km values for B-Raf and MKK1necessitates the development of individual assays for each enzyme ratherthan the coupled assay developed herein. The development of such assayswill undoubtedly facilitate a future understanding of the mode of actionof any inhibitory compounds identified in the screening assay.

For the purposes of defining the ATP load of the screening assay, theconcentration-dependence of the coupled assay was determined. The signalgenerated is maximal and half maximal at ˜200 μM and ˜18 μM ATPrespectively. Further studies indicated that 50 μM was the lowest ATPconcentration at which a robust day-to-day assay signal was observed(e.g. signal: 8000, 2% CV). ATP levels below this (10 μM) resulted in ahigher relative variation in assay signal (e.g. signal: 2500, 10% CV).The final ATP concentration of the screening assay was defined as 50 μMto provide an signal window large enough to support any attrition duringthe transfer of the assay to automation.

Time Course of Screening Assay.

-   -   It is critical that a screening assay is performed within its        period of linearity. To determine the length of the linear phase        of the coupled assay, a time course was performed up to 75        minutes. The reaction was linear between 5 and 45 minutes. The        ‘lag’ period observed is characteristic of this assay format and        reflects the time required to accumulate detectable levels of        reaction product. Based upon this study an incubation period of        45 minutes at room temperature was defined.

Finalised Conditions for B-RafV599E DELFIA Assay

-   -   The summary final screening conditions for the assay were as        follows:

Enzyme Reaction:

-   -   384 well glutathione-coated plate    -   0.05 μl B-Raf lysate    -   6.5 μg/ml GST-MKK1    -   80 μg/ml GST-kdERK2    -   50 μM A TP    -   incubation at room temperature for 45 minutes.    -   Final volume of 25 μl

Detection Conditions:

-   -   1:3000 anti-phosphoERK2 antibody    -   1:1000 Eu-labelled anti-mouse antibody

Automation Development and Quality Control Validation of Screening BatchReagents

-   -   Using the conditions described in above, assays were performed        to compare the screening and assay development batches of B-Raf,        MKKI and ERK-2. In all cases the screening batch of reagents        performed equivalently when compared to those used for assay        development.

Mini-runs of Automated B-RatV599E DELFIA Assay.

In preparation for the screen, an automated liquid handling strategy wasdesigned for the assay. In order to test this system, ‘mini-runs’ of theautomated B-Raf assay were performed using mock screening plates (i.e.no compound but containing standard control columns). The data derivedfrom these experiments represents both a measure of the robustness ofthe biological assay as well as the accuracy and consistency of theautomation processes involved.

In brief, each plate contained B-Raf kinase reactions in all wells ofColumns 1-22 and columns 23-24 contained the control reactions. In orderto define the quality of data generated by the automated assay, 4 platemini-runs using this format were performed on two separate days. Withineach ‘mini run’ one plate was used to define IC50 values for somepredicted inhibitors of this assay. The inhibitors covered a range ofmodes of action: Staurosporine (ATP-competitive kinase inhibitor),SB203580 (ATP-competitive Raf inhibitor) and U0126 (non-ATP competitiveMKK1 inhibitor).

The assay demonstrates good consistency, both within plates and betweendays. The data obtained show that the automated assay achieves thecriteria defined for a 384-format in vitro HTS assay:

-   -   Signal to background of at least 10:1    -   Z′ of >0.4    -   Row and column CVs of <15%

The inhibitors employed further validate the assay by generatingconcentration-dependent inhibition of Raf/MKK1 activity. Of particularimportance is the fact that standard inhibitors returned IC50 valueswithin a 2-fold range on separate days. These data also indicate thatthese compounds would have been identified as hits when tested at ˜30 μm(10 μg/ml) in this screening assay.

Finalised Protocol for Automated B-Raf V599E DELFIA Enzyme Cocktail(Final Volume 12 μl):

0.05 μl Raf lysate

0.0325 μl GST-MKKI 0.065 μl GST-kdERK2

1. 3 μl test compound pre-plated in glutathione-coated 384 plate.2. 12 μl Enzyme cocktail added by PlateMatePlus.3. 10 μl ATP added by Asys.4. Plate shaken at RT for 45 min.5. Plate washed with 3×80 μl/well DELFIA Wash Buffer (DWB) using platewasher ELX405.6. 25 μl of anti-phosphoERK2 added by Multidrop.7. Plate shaken at RT for 1 h8. Plate washed with 3×80 μl/well DWB.9. 25 μl Eu-labelled anti-mouse antibody added by Multidrop.10. Plate shaken for 30 minutes room temperature.11. Plate washed with 3×80 μl/well DWB.12. 25 μl DELFIA Enhancement Solution added by Multidrop.13. Plate incubated at Room Temperature in the dark for 30 minutes.14. Plate read in FUSION.

TABLE 2 This table shows the inhibition of growth of various cell lineswith B-Raf mutations. cell name tissue Ras Mutation B-Raf mutationY13-259 S-phase inhibition SW620 colorectal onc wt inhibited 92% SK-Mel2melanoma wt inhibited 70% HMV11-Riken melanoma wt inhibited 100%  DLD1colorectal K13Asp/wt wt inhibited 40% SW480 colorectal K12 wt inhibited57% LS174T colorectal K12Asp/wt wt inhibited 84% JW2 colorectal K12 wtinhibited 79% CaCO2 colorectal wt wt inhibited 60% HCT-116 colorectalK13 Asp wt inhibited 95% colo741 colorectal V599E not  0% SK-MEL-28melanoma b V599E not  4% WM-266- melanoma b V599D not 10% 4/WM-115 A2058melanoma V599E not  0% Malme melanoma V599E not  0% LS411N colorectalV599E not 0%, 0% HT29 colorectal wt V599E not 15% colo205 colorectal wtV599E not ?3% Mawi colorectal wt V599E not  5% NCI-H2087 NSCLC cell oncL596V inhibited 77% line pair The proportion of inhibition (as apercentage of the number of cells that do not incorporate BrdU) is shownin the last column.

TABLE 3 This table shows the inhibition of cell growth in cells treatedwith the MEK inhibitor U0126. Ras B-Raf U0 UO/ cell name tissue Mutationmutation inhibit S ERK-inhib SW620 colorectal onc wt 92% CHL melanoma wt51% >90% colo741 colorectal V599E 76% 90% SK-MEL-28 melanoma b V599E98% >90% WM-266- melanoma b V599D >99% >90% 4/WM-115 A2058 melanomaV599E 68% 80% NCI-H2087 NSCLC onc L596V 56% >90% cell line pair Mawicolorectal wt V599E 80% >90%

TABLE 4 Inhibition of cell growth by BAY 43-9006. Cell line IC50 μMWM266.4 6.1 Colo 829 5.1 BE 5.4 Cell lines were incubated in thepresence of increasing levels of BAY 43-9006 and the levels of cellgrowth were determined by sulphorhodamine B staining. The IC₅₀ valueswere determined by non-linear regression analysis and are indicated.

TABLE 5 Cell cycle analysis. Proportion of events (%) SiRNA SiRNA SiRNACell line untreated U0126 DMSO BRAF CRAF scrambled Sub-G1 0.8 3.5 0.12.9 0.6 0.3 G1 87.8 87.5 87.8 86.6 86.3 90.1 S 4.8 1.3 4.5 3.6 7.8 3.6G2/M 6.7 7.8 7.5 7 5.4 6.1 WM-266.4 cells were treated with U0126, DMSO,siRNA to BRAF, siRNA to CRAF or the scrambled siRNA control. The cellswere incubated for 96 hours and the cells were fixed and stained withpropridium iodide for cell cycle analysis by FACS. The proportion ofcells in each phase of the cell cycle is shown.

Computational Aspects of Detection

The detection of mutant B-Raf polypeptides and/or mutant B-raf nucleicacids can be automated to provide rapid massively parallel screening ofsample populations. Computerised methods for mutation detection areknown in the art, and will generally involve the combination of asequencing device, or other device capable of detecting sequencevariation in polypeptides or nucleic acids, a data processing unit andan output device which is capable of displaying the result in a forminterpretable by a technician or physician.

In a preferred aspect, therefore, the invention provides an automatedmethod for detecting a mutation at a target sequence position in anucleic acid derived from a naturally-occurring primary human tumourencoding a B-Raf polypeptide, comprising:

-   -   sequencing a sample of an amplification product of the nucleic        acid from the naturally-occurring primary human tumour to        provide a sample data set specifying a plurality of measured        base pair identification data in a target domain extending from        a start sequence position to an end sequence position;    -   determining presence or absence of the mutation in the sample        conditional on whether the measured base pair identification        datum for the target sequence position corresponds to a        reference base pair datum for the target sequence position; and    -   generating an output indicating the presence or absence of the        mutation in the sample as established by the determining step.    -   Methods for sequencing and for detection of mutations in        sequences are set forth above and generally known in the art.        The invention makes use of such methods in providing an        apparatus for carrying out the process of the invention, which        apparatus comprises:    -   a sequence reading device operable to determine the sequence of        a sample of a nucleic acid to provide a sample data set        specifying measured base pair identification data in a target        domain extending from a start sequence position to an end        sequence position; and    -   a data analysis unit connected to receive the sample data set        from the sequencing device and operable to determine presence or        absence of the mutation in the sample conditional on whether the        measured base pair identification datum for the target sequence        position corresponds to a reference base pair datum for the        target sequence position.

Suitable sequence reading devices include automated sequencers,RFLP-analysers and mobility shift analysis apparata. Advantageously, thesequence of an amplification product of the target nucleic acid isanalysed, and the apparatus moreover includes an amplification devicesuch as a PCR machine.

Preferably, the apparatus also comprises an output device operable togenerate an output indicating the presence or absence of the mutation inthe sample determined by the data analysis unit. For example, the outputdevice can comprise at least one of: a graphical user interface; anaudible user interface; a printer; a computer readable storage medium;and a computer interpretable carrier medium.

The invention can moreover be configured to detect the mutant B-Rafprotein itself. Thus, in a further aspect, the invention relates to anautomated method for detecting a single amino acid mutation in a B-Rafpolypeptide from a naturally-occurring primary human tumour, comprising:

-   -   applying a marker to one or more target amino acids in a sample        of the B-Raf polypeptide;    -   reading the sample after applying the marker to determine        presence or absence of the marker in the sample, thereby to        indicate presence or absence of the single amino acid mutation        in the sample; and    -   generating an output indicating the presence or absence of the        single amino acid mutation in the sample as determined by the        reading step.

The marker preferably comprises a ligand that binds differentially to awild-type B-Raf polypeptide without single amino acid mutation and to amutant B-Raf polypeptide with the mutation. Preferential binding toeither form of B-Raf is possible in the context of the invention.

The invention moreover provides an apparatus for detecting an amino acidmutation in a B-Raf polypeptide, comprising:

-   -   a protein marking device loaded with a marker and operable to        apply a marker to one or more target amino acids in a sample of        the B-Raf polypeptide; and    -   a marker reading device operable to determine presence or        absence of the marker in the sample, thereby to indicate        presence or absence of the single amino acid mutation in the        sample.

The marker used can be an antibody, and the protein marking device canbe configured to implement an ELISA process.

Advantageously, the protein marking device comprises a microarrayerwhich is preferably configured to read the sample optically.

Preferably, the apparatus comprises an output device operable togenerate an output indicating the presence or absence of the singleamino acid mutation in the sample as determined by the marker readingdevice. Suitable output devices comprises at least one of: a graphicaluser interface; an audible user interface; a printer; a computerreadable storage medium; and a computer interpretable carrier medium.

Uses of the Invention

The present invention provides novel mutants of B-Raf polypeptides whichare useful in the detection of neoplastic conditions, and thedetermination of prognoses for subjects suffering from such conditions.In general, the presence of a mutation in B-Raf as described herein isassociated with the presence of neoplasia.

In one aspect, the present invention provides a method for identifyingcancerous cells or tissue (such as malignant melanoma, colorectalcancer, breast cancer or NSCLC), or of identifying cells or tissue whichare predisposed to developing a neoplastic phenotype, comprising:amplifying at least part of a B-raf gene of the cells or tissue;analysing the amplification product to detect a mutation in the B-rafgene as described herein; wherein a cell or tissue having one or moreB-raf mutations is categorised as being cancerous or being at anincreased risk of developing a cancerous condition. Suitableamplification means include PCR and cloning.

In another embodiment, the present invention relates to a method fordetermining a prognosis in a subject suffering from cancer (such asmalignant melanoma, colorectal cancer, breast cancer or NSCLC). Themethod comprises: amplifying the region of the B-raf gene as describedabove; analysing the amplification products for evidence of mutation asdescribed above; and classifying a subject having no mutations in theB-raf gene as being less likely to suffer a relapse of the disease aftertherapy and/or surgery, or having an increased chance of survival than apatient having one or more mutations in the region.

The techniques of the invention can also be employed to determine thecourse of therapy to which a subject should be exposed, on the basis ofthe prognosis as set forth above; a subject having a poor prognosis isadvantageously handled using a more aggressive therapy that a subjecthaving a good prognosis.

The techniques according to the invention can be automated, as requiredfor rapid screening of samples for the identification of potentiallycancerous conditions. Generally, an automated process will compriseautomated amplification of nucleic acid from tissue or cell samples,detection of mutations in amplified nucleic acid, such as by fluorescentdetection, and/or displaying the presence of mutations. Exemplaryautomated embodiments are described above.

The identification of mutant B-Raf according to the invention can thusbe used for diagnostic purposes to detect, diagnose, or monitordiseases, disorders, and/or conditions associated with the expression ofmutant B-Raf. In particular, the invention is concerned with thedetection, diagnosis and/or monitoring of cancers associated with mutantB-Raf as set forth herein.

The invention provides a diagnostic assay for diagnosing cancer,comprising (a) assaying the expression of mutant B-Raf in cells or bodyfluid of an individual using one or more antibodies specific to theB-Raf mutant as defined herein. The presence of mutant B-raf transcriptin biopsy tissue from an individual can indicate a predisposition forthe development of the disease, or can provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type allows health professionals to employpreventative measures or aggressive treatment earlier thereby preventingthe development or further progression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., (1985) J. Cell.Biol. 101:976-985; Jalkanen, et al., (1987) J. Cell. Biol.105:3087-3096). Other antibody-based methods useful for detectingprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I),carbon (¹⁴C), sulphur (³⁵S), tritium (³H), indium (¹¹²In) and technetium(⁹⁹Tc); luminescent labels, such as luminol; and fluorescent labels,such as fluorescein and rhodamine, and biotin.

Moreover, mutations in B-raf can be detected by analysis of nucleicacids, as set forth herein. For example, the presence of mutations canbe detected by sequencing, or by SCCP analysis.

The present invention moreover provides kits that can be used in theabove methods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody can be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognises the first antibody can be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificfor mutant B-Raf polypeptides as described herein. Such a kit caninclude a control antibody that does not react with the mutant B-Rafpolypeptide. Such a kit can include a substantially isolated polypeptideantigen comprising an epitope which is specifically immunoreactive withat least one anti-B-Raf antibody. Further, such a kit includes means fordetecting the binding of said antibody to the antigen (e.g., theantibody can be conjugated to a fluorescent compound such as fluoresceinor rhodamine which can be detected by flow cytometry). In specificembodiments, the kit can include a recombinantly produced or chemicallysynthesised polypeptide antigen. The polypeptide antigen of the kit canalso be attached to a solid support.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the mutant B-Rafpolypeptide of the invention. The diagnostic kit includes asubstantially isolated antibody specifically immunoreactive withpolypeptide or polynucleotide antigens, and means for detecting thebinding of the polynucleotide or polypeptide antigen to the antibody. Inone embodiment, the antibody is attached to a solid support. In aspecific embodiment, the antibody can be a monoclonal antibody. Thedetecting means of the kit can include a second, labelled monoclonalantibody. Alternatively, or in addition, the detecting means can includea labelled, competing antigen.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are apparent to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1. A mutant human B-Raf polypeptide comprising one or more amino acidmutations.
 2. A mutant according to claim 1, wherein the single aminoacid mutations are located C-terminal to amino acid 300 in B-Raf.
 3. Amutant according to claim 2, wherein the amino acid mutations occur atone or more of positions 463, 465, 468, 585, 594, 595, 596 and 599 inB-Raf.
 4. A mutant according to claim 1, wherein the single amino acidmutations are selected from the group consisting of V599E, V599D, G595R,G465V, G465E, G465A, G468A, G468E, E585K, F594L, G595R, L596V, L596R andG463E. 5-7. (canceled)
 8. A ligand which binds selectively to apolypeptide according to claim
 1. 9. A ligand according to claim 8 whichis an immunoglobulin.
 10. A ligand according to claim 9, which is anantibody or an antigen-binding fragment thereof.
 11. A method for thedetection of oncogenic mutations, comprising the steps of: (a) isolatinga sample of naturally-occurring cellular material from a human subject;(b) examining nucleic acid material from at least part of one or moreB-raf genes in said cellular material; and (c) determining whether suchnucleic acid material comprises one or more point mutations in asequence encoding a B-Raf polypeptide.
 12. A method for the detection ofoncogenic mutations, comprising the steps of: (a) isolating a firstsample of cellular material from a naturally-occurring tissue of asubject which is suspected to be cancerous, and a second sample ofcellular material from a non-cancerous tissue of the same subject; (b)examining nucleic acid material from at least part of one or more B-rafgenes in both said samples of cellular material; and (c) determiningwhether such nucleic acid material comprises one or more point mutationsin a sequence encoding a RAF polypeptide; and said mutation beingpresent in the naturally-occurring cellular material from the suspectedcancerous tissue but not present in the cellular material from thenon-cancerous tissue.
 13. A method according to claim 11 or claim 12,wherein the point mutation occurs at one or more of positions 1388,1394, 1403, 1753, 1782, 1783, 1796, 1797, 1787 and 1786 of B-raf.
 14. Amethod according to claim 13, wherein the point mutation is G1388T,G1783C, TG1796-97AT, G1394T, G1394A, G1394C, G1403C, G1403A, G1753A,T1782G, G1388A, T1796A, T1787G or C1786G in B-raf.
 15. A method for thedetection of oncogenic mutations, comprising the steps of: (a) obtaininga sample of cellular material from a subject; (b) screening said samplewith a ligand according to claim 13; and (c) detecting one or moremutant B-Raf polypeptides in said sample.
 16. A method according toclaim 15, wherein the mutant B-Raf polypeptide is a polypeptideaccording to claim
 1. 17. Apparatus for detecting a mutation at a targetsequence position in a nucleic acid encoding a B-Raf polypeptide,comprising: a sequence detecting device operable to monitor the sequencea sample of an amplification product of the nucleic acid to provide asample data set specifying measured base pair identification data in atarget domain extending from a start sequence position to an endsequence position; and a data analysis unit connected to receive thesample data set from the sequencing device and operable to determinepresence or absence of the mutation in the sample conditional on whetherthe measured base pair identification datum for the target sequenceposition corresponds to a reference base pair datum for the targetsequence position.
 18. The apparatus of claim 17, an output deviceoperable to generate an output indicating the presence or absence of themutation in the sample determined by the data analysis unit.
 19. Theapparatus of claim 18, wherein the output device comprises at least oneof: a graphical user interface; an audible user interface; a printer; acomputer readable storage medium; and a computer interpretable carriermedium.
 20. An automated method for detecting a mutation at a targetsequence position in a nucleic acid encoding a B-Raf polypeptide,comprising: sequencing a sample of an amplification product of thenucleic acid to provide a sample data set specifying measured base pairidentification data in a target domain extending from a start sequenceposition to an end sequence position; determining presence or absence ofthe mutation in the sample conditional on whether the measured base pairidentification datum for the target sequence position corresponds to areference base pair datum for the target sequence position; andgenerating an output indicating the presence or absence of the mutationin the sample as established by the determining step.
 21. Apparatus fordetecting a single amino acid mutation in a B-Raf polypeptide,comprising: a protein marking device loaded with a marker and operableto apply a marker to one or more target amino acids in a sample of theB-Raf polypeptide; and a marker reading device operable to determinepresence or absence of the marker in the sample, thereby to indicatepresence or absence of the single amino acid mutation in the sample. 22.The apparatus of claim 21, wherein the marker comprises a ligand thatbinds preferentially to a B-Raf polypeptide bearing the single aminoacid mutation.
 23. The apparatus of claim 21, wherein the markercomprises a ligand that binds preferentially to a B-Raf polypeptide of awild-type without the single amino acid mutation.
 24. The apparatus ofclaim 21, wherein the marker is an antibody.
 25. The apparatus of claim21, wherein the protein marking device is configured to implement anELISA process.
 26. The apparatus of claim 21, wherein the proteinmarking device comprises a microarrayer.
 27. The apparatus of claim 21,wherein the marker reading device is configured to read the sampleoptically.
 28. The apparatus of claim 21, comprising an output deviceoperable to generate an output indicating the presence or absence of thesingle amino acid mutation in the sample as determined by the markerreading device.
 29. The apparatus of claim 28, wherein the output devicecomprises at least one of: a graphical user interface; an audible userinterface; a printer; a computer readable storage medium; and a computerinterpretable carrier medium.
 30. An automated method for detecting asingle amino acid mutation in a B-Raf polypeptide, comprising: applyinga marker to one or more target amino acids in a sample of the B-Rafpolypeptide; reading the sample after applying the marker to determinepresence or absence of the marker in the sample, thereby to indicatepresence or absence of the single amino acid mutation in the sample; andgenerating an output indicating the presence or absence of the singleamino acid mutation in the sample as determined by the reading step. 31.A method for identifying one or more compounds having anti-proliferativeactivity, comprising the steps of: (a) providing one or more mutantB-Raf polypeptides in accordance with the present invention; (b)contacting said polypeptide(s) with one or more compounds to be tested;and (c) detecting an interaction between said one or more compounds andsaid mutant polypeptides.
 32. A method according to claim 31, whereinthe interaction is a binding interaction.
 33. An assay for identifyingone or more compounds having anti-proliferative activity, comprising thesteps of: (a) providing one or more mutant B-Raf polypeptides inaccordance with the present invention; (b) providing a downstreamsubstrate for the B-Raf polypeptide; (c) detecting modification of thesubstrate in presence of the compound(s) to be tested.
 34. An assayaccording to claim 33, wherein the substrate modification is detecteddirectly.
 35. An assay according to claim 34, wherein the substrate isan enzyme which modifies a second substrate, which second modificationis detectable.
 36. An assay according to claim 35, wherein the substrateis MEK and the second substrate is MAPK.
 37. A method or assay accordingto claim 21 or 33, wherein a reference level is determined for the assayin absence of the compound or compounds to be tested.
 38. Aconstitutively active kinase comprising a mutation in the phosphatebinding loop thereof selected from the group consisting of mutations atone or more positions corresponding to positions 463, 465 and 468 ofB-Raf.
 39. A kinase according to claim 38, wherein the mutation is atone or more of positions 463 and
 468. 40. A kinase according to claim39, wherein the mutation is selected from the group consisting of G463Vand G468A.
 41. A kinase according to claim 38, which is a Raf proteinkinase.
 42. A kinase according to claim 41 which is B-Raf.
 43. A methodfor screening one or more compounds for an inhibitory effect on akinase, comprising: (a) preparing a mutant kinase comprising an aminoacid substitution, deletion or insertion according to claim 38; (b)exposing the mutant kinase to said one or more compounds in the presenceof a kinase substrate; and (c) determining the ability of the kinase tophosphorylate the substrate in the presence of the one or morecompounds.